translocation of transition metal oxide nanoparticles to breast...

Contents lists available at ScienceDirect

Environment International

journal homepage: www.elsevier.com/locate/envint

Translocation of transition metal oxide nanoparticles to breast milk andoffspring: The necessity of bridging mother-offspring-integrationtoxicological assessments

Jie Cai, Xinwei Zang1, Zezhong Wu1, Jianxin Liu, Diming Wang⁎

College of Animal Sciences, Dairy Science Institute, MOE Key Laboratory of Molecular Animal Nutrition, Zhejiang University, Hangzhou 310029, PR China

A R T I C L E I N F O

Handling Editor: Olga-Ioanna Kalantzi

Keywords:NanoparticlesToxicologyLactationMammary glandBreastfeedingOffspring

A B S T R A C T

Although infant nanomaterial exposure is a worldwide concern, breastfeeding transfer of transition metal-oxidenanoparticles to as well as their toxicity to offspring are still unclear. Breastfeeding transmits nutrition andimmunity from mothers to their offspring; it also provides a portal for maternal toxins to enter offspring. Thus, atoxicology assessment of both mothers and their offspring should be established to monitor nanomaterial ex-posure during lactation. Here, we determined the effects of the exposure route on the biodistribution, bio-persistence, and toxicology of nanoparticles (titanium dioxide, zinc oxide, and zirconium dioxide) in both mousedams and their offspring. Oral and airway exposure routes were tested using gavage and intranasal adminis-tration, respectively. Biodistribution in the main organs (breast, liver, spleen, lung, kidney, intestine, and brain)and biopersistence in the blood and milk were determined using inductively coupled plasma mass spectrometry.Hematology and histomorphology analyses were performed to determine the toxicology of the nanoparticles. Areduced offspring body weight was found with the reduced nanoparticle size. Furthermore, both oral and airwayexposure increased the nanoparticle concentrations in the main tissues and milk. More nanoparticles weretransferred into maternal tissues and milk via airway exposure than via oral exposure. During the transfer of themetal from the exposed nanoparticles to milk, the immune cell pathway played a more important role in theairway route than in the oral exposure route. Finally, maternal exposure via both the oral and airway routesreduced the body weight and survival rate of their breastfeeding offspring, which could possibly be attributed tothe toxicity of nanoparticles to blood cells and organs. In conclusion, maternal exposure to nanoparticles led to areduced body weight and survival rate in breastfed offspring, and nanoparticle exposure via the airway route ledto a higher immune response and tissue injury than that via the oral exposure route. This study suggests that theuse of products containing metal nanoparticles in breastfeeding mothers and their offspring should be recon-sidered to maintain a safe breastfeeding system.

1. Introduction

The postnatal and early childhood period is a window of specialvulnerability, in which exogenous hazards can threaten children'shealth. In 2015,> 156 million growth-retarded children were born andwere likely to have higher risk for chronic diseases (such as diabetes

and heart disease) (Black et al., 2013; Stewart et al., 2013; UNICEFet al., 2016). One of the biggest causes of these growth-retarded chil-dren is environmental exposure to toxins in utero (UNICEF et al., 2016).Most studies focused on the traditional risks for the origin of growthretardation, whereas emerging risks (such as new chemicals and toxicsubstances) are underestimated. Currently, nanomaterials are

https://doi.org/10.1016/j.envint.2019.105153Received 7 June 2019; Received in revised form 3 September 2019; Accepted 3 September 2019

Abbreviations: %BASO, basophil percentage; %EOS, eosinophil percentage; %LYM, lymphocyte percentage; %MONO, monocyte percentage; %NEU, neutrophilpercentage; %RETIC, reticulocyte percentage; AUC, area under the curve; BASO, basophil count; BW, body weight; ED, experiment day; EOS, eosinophil count; HCT,hematocrit; HGB, hemoglobin concentration; LYM, lymphocyte count; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration;MCV, mean corpuscular volume; MONO, monocyte count; MPV, mean platelet volume; NEU, neutrophil count; PBS, phosphate buffered saline; PCT, plateletcrit;PDW, platelet distribution width; PLT, platelet count; RBC, red blood cell count; RDW, red cell distribution width; RETIC, reticulocyte count; SEM, scanning electronmicroscopy; TEM, transmission electron microscope; TiO2, titanium dioxide; WBC, white blood cell count; ZnO, zinc oxide; ZrO2, zirconium dioxide

⁎ Corresponding author.E-mail addresses: [email protected] (J. Cai), [email protected] (X. Zang), [email protected] (J. Liu), [email protected] (D. Wang).

1 Equal contribution

Environment International 133 (2019) 105153

0160-4120/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

T

http://www.sciencedirect.com/science/journal/01604120

https://www.elsevier.com/locate/envint

https://doi.org/10.1016/j.envint.2019.105153


mailto:[email protected]





http://crossmark.crossref.org/dialog/?doi=10.1016/j.envint.2019.105153&domain=pdf

increasingly applied in foods, medicines, cosmetics and electronics dueto their physicochemical properties and novel functions (Stark et al.,2015). The widespread use of nanomaterials has led to potential healthissues in children, such as neurodevelopmental retardation, metabolicdisorders, immune diseases and growth retardation (Ema et al., 2016;Ema et al., 2017; Hougaard et al., 2015).

The entrance of nanomaterials into neonates is controllable by re-stricting the direct exposure of nanomaterials from baby formulathrough a nanofood-labeling regime, which was implemented by theEuropean Union and recommended by the FDA in the US (Illuminatoand Friends of the Earth U.S., 2016); however, nanomaterials fromdrugs, cosmetics, electronics and the air (both in the workplace andoutdoors) are still accessible to the mother and are potential toxicthreats to offspring by indirect exposure through breastfeeding transfer.Breastfeeding transmits nutrition and immunity from the mother to theoffspring (Nabhan, 2015), which helps prevent various diseases inoffspring (Victora et al., 2016). Child mortality data worldwide (2000)suggested that exclusive breastfeeding is the most effective way to in-crease the survival rate of children under 5 years old (Jones et al.,2003), indicating the importance of breastfeeding to an infant's healthstatus. However, toxins transferred into milk from exogenous sources,such as heavy metals (Ou et al., 2018; Sharma et al., 2019), mycotoxins(Braun et al., 2018), and organic halogens (Čechová et al., 2017; Taoet al., 2017), have become a sizeable issue for breastfeeding neonatesthat has been drawing attention worldwide (Landrigan et al., 2002;Lehmann et al., 2014). Although lactating women are cautious inchoosing their daily products and food, nanomaterials such as titaniumdioxide (TiO2), zinc oxide (ZnO), and zirconium dioxide (ZrO2) arealready present in foods, cosmetics, toothpaste, drugs, and bio-transplants (Anklam et al., 2011; Deepthi et al., 2016). A substantialconcern remains whether a breastfeeding mother exposed to nanoma-terials will deliver milk with nanoparticles to their offspring(Mohammadipour et al., 2013; Shimizu et al., 2009). Previous studies inrats have revealed the presence of nanoparticles in breast milk (Melniket al., 2013; Yang et al., 2018). However, these studies failed to describehow the nanoparticles were transferred, as the lactating breast offers acomplex transfer system, including exocytotic, lipid secretion, trans-cytotic, membrane transport, and paracellular transport pathways(Fleishaker, 2003; McManaman and Neville, 2003). Moreover, severalstudies in rats or mice attempted to determine how nanoparticlestransferred via breast milk could affect the health status of their off-spring, but the toxicological assessment data of offspring was notavailable (Melnik et al., 2013; Morishita et al., 2016; Sumner et al.,2010). Thus, a systematic evaluation of the breastfeeding transfer oftransition metal-oxide nanoparticles and their toxicological effects onboth the mother and offspring is needed.

In this study, we determined the effects of oral and airway exposureto nanoparticles with different physicochemical properties based on thefollowing: 1) the presence and transfer of nanoparticles in milk; 2)breast structure and lactation performance; 3) the distribution of na-noparticles delivered by breastfeeding in offspring's tissue; and 4) atoxicology assessment of nanoparticle-exposed dams and their off-spring. TiO2, ZnO, and ZrO2 nanoparticles were chosen not only be-cause of their potential exposure risk for the lactating mother (Haoet al., 2017; Mohammadipour et al., 2013) but also because of theirsimilarities and differences in chemical characteristics (Zr and Ti arehomologous, while Ti and Zn are synperiodic) would give a glimpse ofhow transition metal oxide nanoparticles are delivered from mother tooffspring and potentially impact the health of mothers and their off-spring.

2. Materials and methods

2.1. Physicochemical characterization of nanoparticles

Powder-form nanoparticles of TiO2 (anatase, molecular weight:

79.9), ZnO (molecular weight: 81.4), and ZrO2 (molecular weight:123.2) with diameters of 100 nm (n100TiO2, n100ZnO, and n100ZrO2),50 nm (n50TiO2, n50ZnO, and n50ZrO2), and 10 nm (n10TiO2,n10ZnO, and n10ZrO2), were obtained from Zhonghang NanometerTechnology (Hefei, China). The morphology and approximate diameterof the powder-form nanoparticles was determined by scanning electronmicroscopy (SEM) using a HITACH-4800S microscope (Hitachi, Japan).The size distribution spectrum, average particle size and zeta potentialof each type of nanoparticle were measured by a Zetasizer Nano-ZS(Malvern Instruments, Worcestershire, UK) after the powder-form na-noparticles were suspended in double deionized water and dispersed(0.5 mmol/L). The specific surface area of the nanoparticles was mea-sured by nitrogen adsorption−desorption analysis (at −196 °C) usingthe ASAP 2020 instrument (Micromeritics, Norcross, Georgia, USA).The physicochemical characterization of the nanoparticles, includingthe average particle size, zeta potential and pH, was also determinedusing their serum suspensions. Prior to the analysis, the serum sus-pension of the nanoparticles was prepared by diluting a water suspen-sion of the nanoparticles with 10% fetal calf serum (Macklin, Australia)to 0.5mmol/L. The pH values of the nanoparticle suspensions weremeasured by a pH meter (Mettler Toledo, Beaumont Leys, Leicester,UK).

2.2. Animal experiments

All procedures involving animal experiments were approved by theAnimal Use and Care Committee of Zhejiang University (Hangzhou,China). Pregnant ICR mice (12–13 weeks, gestation day: 14–16) werepurchased from the Shanghai Experimental Animal Center of theChinese Academy of Sciences (Shanghai, China). Pregnant mice (laterlactating mice) and their offspring were raised in a room at 25 °C with12 h light/dark cycles. The standard basal food and water were given adlibitum via food pellets and water supply systems, respectively. Lactatingmice were exposed to the nanoparticles between lactating days 1 [ex-periment day (ED) 1] and 21. To select an effective nanoparticle sizethat is potentially toxic to maternal mice and their offspring, lactatingmice were exposed to all sizes of the different nanoparticles (n100TiO2,n100ZnO, and n100ZrO2; n50TiO2, n50ZnO, and n50ZrO2; n10TiO2,n10ZnO, and n10ZrO2) through the oral route (0.2 mmol/kg, far lowerthan the half-lethal dose at their normal size). Lactating mice wereorally administered the nanoparticle solutions (140 μL) or saline bygavage every day. For airway exposure to the nanoparticles (0.2 mmol/kg), lactating mice were anesthetized with isoflurane and intranasallyadministered the nanoparticle solutions or saline in 10 separate ad-ministrations (7 μL for each nostril in each administration) within 2 hevery day. The body weight (BW) of dams was recorded over the entirelactation period (ED 1, ED 7, ED 14, and ED 21). The BW of offspringwas recorded from postnatal day (ED 1) 1 to 42 (maturity) every weekand were summed to yield the litter weight. Normally, offspring wereweaned at postnatal day 21 (ED 21). Some offspring (4 from eachgroup) that consumed milk from dams with nanoparticle exposure wereweaned early at postnatal day 14 (ED 14) and were weighed from ED 14to ED 42 every week to verify the growth retardation effect of nano-particles in milk. These early-weaned offspring were separated fromtheir mothers and fed ad libitum for a week by the water supply systemwith a mimic mouse breast-feeding milk made using commercial animalmilk powder, which mainly contained 8% crude protein, 2.1% crudefat, and 4% carbohydrate. The survival rate of offspring was recordedon the same days that the BW was recorded.

2.3. Nanoparticle solutions prepared for administration

For oral or airway administration, n10TiO2, n50TiO2, n100TiO2,n10ZnO, n50ZnO, n100ZnO, n10ZrO2, n50ZrO2, and n100ZrO2 wereweighed accurately and diluted with a sterile saline solution to50mmol/L. The nanoparticle solutions were dispersed using an

J. Cai, et al. Environment International 133 (2019) 105153

2

(caption on next page)


3

ultrasonic bath (GT SONIC, Guangdong, China) at a 40 kHz frequencyfor 30min before oral or airway administration.

2.4. Blood collection

Blood was collected from the submandibular vein. For the nano-particle hemodynamic analysis of dams, blood samples (50 μL) weretaken at 0.25, 0.5, 0.75, 1, 2, 4, 8, and 12 h after treatment on ED 1, ED6, and ED 11, respectively. The blood samples collected on the sametime point of ED1, ED6, and ED11 were pooled proportionally. Bloodsamples (100 μL) taken from dams on ED 14 were used for a hema-tology analysis. Blood samples taken from offspring on ED 14 were usedfor a hematology analysis (100 μL) and blood nanoparticle concentra-tion analysis (50 μL). Blood samples (50 μL) taken on ED 21, ED 22, ED24, ED 28, ED 35, and ED 42 from dams and offspring were used tocalculate the elimination rates and half-lives of the nanoparticles in theblood, respectively.

2.5. Milk collection

Milk samples were collected from the mammary gland 30 s after thesubcutaneous injection of oxytocin (1.8 U/mouse, Kassandra et al.,2014). To obtain skim milk, a portion of the milk samples (100 μL) wascentrifuged for 15min (2000×g, 4 °C), and the top layer, which con-tained the milk fat globules, was removed; it was then filtered through asyringe filter to remove the remaining supernatant. For dynamic ana-lysis of the nanoparticles in the milk of dams, milk samples (50 μL) weretaken at 0.25, 0.5, 0.75, 1, 2, 4, 8, and 12 h after treatment on ED 1, ED6, and ED 11, respectively. The milk samples collected on the same timepoint of ED1, ED6, and ED11 were pooled proportionally. To determinethe milk composition [including total protein, fat, lactose, β-casein,milk serum albumin, IgA, somatic cell (SC) count, and reactive oxygenspecies (ROS)], milk samples (100 μL) were taken on ED 10 and ED 11(peak lactation stage).

2.6. Tissue collection

After the sacrifice of mice by CO2 inhalation, the mammary glandtissues of lactating dams were collected from a subset of dams (n=3)on ED 12 to determine the mammary gland structure and viability usingan immunohistochemical analysis and immunofluorescent staining. Thedata of dams from which tissue collection was performed and theiroffspring were not recorded in the subsequent experiments. Tissuesamples (liver, spleen, lungs, kidneys, intestine, brain, and mammarygland) were collected from the remaining dams on ED 21 to determinethe nanoparticle concentrations. The liver, spleen, lungs, kidneys, in-testine, brain, and mammary gland of offspring were collected on ED 21to determine the nanoparticle concentrations, and HE staining wasperformed (HE staining was not performed on offspring’ mammaryglands because there would be no accumulation of nanoparticles in themammary gland).

2.7. Hematology analysis

Blood samples taken within 2 h were measured by a hematologyautoanalyzer (IDEXX Laboratories, Inc., Westbrook, Maine, USA). Thetested hematological indices included red blood cell count (RBC), he-matocrit (HCT), hemoglobin concentration (HGB), mean corpuscularvolume (MCV), mean corpuscular hemoglobin (MCH), mean

corpuscular hemoglobin concentration (MCHC), red cell distributionwidth (RDW), reticulocyte count (RETIC), white blood cell count(WBC), neutrophil count (NEU), lymphocyte count (LYM), monocytecount (MONO), eosinophil count (EOS), basophil count (BASO), re-ticulocyte percentage (%RETIC), neutrophil percentage (%NEU), lym-phocyte percentage (%LYM), monocyte percentage (%MONO), eosi-nophil percentage (%EOS), basophil percentage (%BASO), plateletcount (PLT), mean platelet volume (MPV), platelet distribution width(PDW), and plateletcrit (PCT).

2.8. Milk yield

The milk yield was determined by offspring weights and weightgains during the peak lactation period (ED 10 and ED 11) according tothe previous method (Sampson and Jansen, 1984). In brief, each day(24 h) was divided to continuous 6 h. During each period, mother andoffspring were separated for 4 h before re-uniting suckling intervals(2 h). Daily milk yield was determined based on offspring weight gainduring 4 suckling intervals for each day and were further corrected withoffspring weight loss.

2.9. HE staining

The tissues samples were fixed in a 4% paraformaldehyde solution(Servicebio, Wuhan, China) for 4 h, and then embedded in paraffinblocks. Sectioned into 4 μm, the samples were deparaffinized and hy-drated by immersion in dimethylbenzene (Sinopharm ChemicalReagent Co., Ltd., Shanghai, China) twice (20min/time), in 100%ethanol (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) twice(5min/time), and then in 75% ethanol (Sinopharm Chemical ReagentCo., Ltd., Shanghai, China) for 5min. The slices were washed by waterand immersed in a hematoxylin staining solution for 5min. The sliceswere washed by water and differentiated with a differentiation solution(Servicebio, Wuhan, China). After washing with water, the slices werestained with bluing fluid (Servicebio, Wuhan, China). After washingwith water, the slices were dehydrated by immersion in 85% ethanol(Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) for 5min and95% ethanol (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China)for 5min. After staining by eosin for 5min, the slices were dehydratedby 100% ethanol (Sinopharm Chemical Reagent Co., Ltd., Shanghai,China) 3 times (5min/time) and transplanted by dimethylbenzene(Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) twice(20min/time), and then mounted on neutral balsam.

2.10. Immunohistochemical analysis

The mouse mammary glands were sampled and fixed in a 4% par-aformaldehyde solution (Servicebio, Wuhan, China) for 4 h prior tobeing embedded in paraffin blocks. Sectioned into 4 μm, the sampleswere deparaffinized and hydrated (detailed information is described in“HE staining”). Antigen retrieval was conducted by boiling the slices incitric acid antigen retrieval buffer (pH=6.0, Servicebio, Wuhan,China) for 8min. The slices were cooled for 8min and heated again for7min. After cooling to room temperature, the slices were washed byphosphate buffered saline (PBS, pH=7.4) on the decoloration shaker 3times (5min each) and immersed in 3% aqueous hydrogen peroxide for25min and washed again. The slices were incubated with 3% BSA(Servicebio, Wuhan, China) for 30min and exposed to primary anti-bodies (anti-claudin-3, 1:400, Proteintech, Chicago, USA) overnight at

Fig. 1. Milk yield and milk composition of lactating dams with nanoparticle exposure in oral or airway route. (a) The milk yield was measured during the peak milkproduction period (lactating days 10 and 11). -A represents airway exposure, eO represents oral exposure. n10, n50, and n100 represent material particle sizes of10 nm, 50 nm, and 100 nm, respectively. (b) The milk composition (total protein, fat, lactose, β-casein, milk serum albumin, IgA, somatic cell count, and reactiveoxygen species) was measured during the peak lactation stage. (c) The percentage of different milk cells was measured during the peak lactation stage. The data arepresented as the mean ± standard error (n=9). * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001.


4



5

4 °C. After washing, the slices were incubated with secondary anti-bodies (HRP-labeled goat anti-rabbit, 1:200, Servicebio, Wuhan, China)at room temperature for 50min and colored by diaminobenzidine(Servicebio, Wuhan, China). The slices were counterstained by hema-toxylin (Servicebio, Wuhan, China).

The staining intensity of claudin-3 in the mammary gland for eachsample was quantified by ImageJ software (IJ 1.46r) and corrected bythe cell numbers. For each slice under the microscope, 6 visual fieldsaround the parts of mammary acini were selected to measure thestaining intensity and cell number. The corrected staining intensity wascalculated by the following equation: corrected staining in-tensity= total staining intensity in the selected area/cell number.

2.11. Immunofluorescence TUNEL apoptosis analysis

The mouse mammary glands were sampled, fixed in a 4% paraf-ormaldehyde solution (Servicebio, Wuhan, China) for 4 h, embedded inparaffin blocks and then sectioned into 4 μm. The paraffin sections weredeparaffinized and hydrated (detailed information is described in “HEstaining”). The dewaxed sections were restored by incubation withproteinase K at 37 °C for 25min and washed with PBS (pH=7.4) 3times (5min). The sections were incubated with a membrane ruptureliquid at 25 °C for 20min and washed with PBS (pH=7.4) 3 times(5min). The apoptotic response of each section was detected by aTUNEL kit (Roche) with a mixture of terminal deoxynucleotidyltransferase and dUTP (mixed ratio was 1:9) at 37 °C for 2 h. Each sec-tion was counterstained by DAPI. The TUNEL-positive nuclei (FITCstained, green color) and TUNEL-negative nuclei (DAPI stained, bluecolor) were counted for each section. The apoptotic index of eachsection was calculated by the percentage of TUNEL-positive nuclei re-lative to the total number of nuclei.

2.12. Localization of nanoparticles in the mammary gland

The mammary gland samples were taken and fixed immediatelywith 2.5% glutaraldehyde. After being fixed overnight, the sampleswere washed with phosphate buffer and further fixed with 1% osmiumtetroxide for 2 h. The samples were dehydrated with a graded series ofethanol solutions (30%, 50%, 70%, 80%, 90% and 95%) for 15min ateach step and then dehydrated with 100% ethanol for 20min. Afterinfiltration with propylene oxide, the samples were embed in the resinand sliced into ultrathin sections (75 nm), which were mounted oncopper grids. The observation of the samples was performed by atransmission electron microscope (TEM, Hitachi Model H-7650,Hitachi, Japan). The presence of nanoparticles was confirmed by energydispersive X-ray spectroscopy.

2.13. Localization of nanoparticles in milk

The precipitate obtained during the milk centrifugation was col-lected, washed with PBS (twice) and suspended in 100 μL PBS toquantify the exposed material in the milk cells. The cell number in thecell suspension was calculated by an Automated Cell Counter (ThermoFisher Scientific, Waltham, MA, US). The exposed materials in the ob-tained skim milk were determined by ICP-MS. For further analysis ofthe exposed material in each milk cell type, flow cytometry sorting wasused (Hassiotou et al., 2013). A cell suspension was obtained fromanother milk sample after centrifugation for 15min (200×g, 4 °C) and

was incubated for 15min at 25 °C with fluorescent-labeled antibodiesCD24, CD45, CD11b, Ly6G, and F4/80 (Supplementary Table S1). Thecells were washed twice in PBS and incubated for 15min at 25 °C withDAPI (Supplementary Table S1). The cells were analyzed and sortedwith a Flow Cytometry Sorter (Becton Dickinson, Franklin Lakes, NJ,USA). The gating strategy is described in the supplementary material(Supplementary Fig. S1). Respective isotype controls were used tostandardize the background fluorescence (Supplementary Table S1).The exposed materials in the sorted milk cells were determined by ICP-MS.

2.14. Determination of exposed material in biosamples

The samples of tissues, milk, milk cells, and whole blood were dis-solved in the digestion solution (HNO3, 65%–68%, GR, SinopharmChemical Reagent, Beijing, China) while heating at 200 °C. The tem-perature was increased to 280 °C when the reaction reached equili-brium and held there for 2.5 h. The digested samples were filtered witha syringe-driven filter (0.22 μm) and diluted with double deionizedwater after removal from the heating block. Then, the material con-centrations in the samples were determined with inductively coupledplasma mass spectrometry (ICP-MS, PerkinElmer NexION 300×,Houston, Texas, USA). The calibration curves (R2 > 0.9999) of 47Tiand 90Zr were generated by ICP-MS with standard solutions of 1, 5, 50,and 200 ng/mL, and the calibration curve (R2 > 0.999) of 66Zn wasgenerated by ICP-MS with standard solutions of 1, 5, 20, 50, and200 ng/mL; these curves were used to determine the respective con-centrations of the nanoparticles in the dam and offspring samples. Thelimits of detection for Ti, Zn, and Zr were confirmed as 0.037, 0.083,and 0.002 ng/mL, respectively, before analysis of the biosamples.

2.15. Calculations

The area under the curve (AUC) values for the dynamic nanoparticleconcentrations in milk and blood were calculated and corrected withthe control group values to eliminate the background nanoparticleconcentration. The AUC values for milk and blood were calculatedusing the trapezoidal rule, following the equation:

∑= ⎡⎣⎢

⎛⎝

+−

+ ⎞⎠

− − ⎤⎦⎥=

− − −y y g gAUC

2 2(xi xi 1) ,

i

n i i i i1

1 1 1

= = … −iy f(x ), 1, 2, ,n 1,i i

= = … −g g(x ), i 1, 2, ,n 1,i i

where n represents the number of sampling times, f(xi) represents thefunction for the material concentration in the treatment group, and g(xi) represents the function for the material concentration in the controlgroup.

The elimination rate (k) was calculated using a regression analysisof the nanoparticle blood concentration versus time. The half-lives ofnanoparticles were calculated by ln 2/k.

The rates of BW change were calculated using the following equa-tion: Rate of BW change= present BW – BW of the start day (g)/BW ofthe start day (g)× 100%.

2.16. Statistics

The comparisons of results between the oral administration groups

Fig. 2. Size-dependent effect of nanoparticles on maternal body weight changes, offspring body weight and offspring survival rate. After dams were exposed todifferent nanoparticles of all sizes orally, the maternal body weight change (a), offspring body weight (b) and offspring survival rate (c) were measured weekly. Thedata are presented as the mean ± standard error for the maternal body weight change and offspring body weight (a, b: n=9 for week 1 after parturition, and n=6for week 2 and week 3 after parturition). The survival rate data is presented as the total survival rate for a certain group. * represents P < 0.05, and *** representsP < 0.001. -A represents airway exposure, eO represents oral exposure. n10, n50, and n100 represent material particle sizes of 10 nm, 50 nm, and 100 nm,respectively.


6

Fig. 3. Entrance of the nanoparticles into the blood circulation system and translocation of the nanoparticles into breast milk after lactating dams were exposed tonanoparticles. After the dams were exposed to nanoparticles in oral or airway route, the concentrations of Ti, Zn, and Zr in blood (a) and milk (b) were measured inthe respective nanoparticle exposure group and control group on experiment day 1, 6 and 11. The area under the curve (c) for blood and milk as well as the milk/blood ratios for Ti, Zn, and Zr were calculated based on the data presented in (a) and (b). -A represents airway exposure, eO represents oral exposure. The data arepresented as the mean ± standard error for (a) and (b) (n= 9) and as a single calculation value for (c).


7



8

and oral control group or between the airway administration groupsand the airway control group were carried out by one-way analysis ofvariance (ANOVA) and the L.S.D. test using SAS (version 9.21, Cary,North Carolina, USA). The comparisons of results between groups withthe oral administration of certain nanoparticles and the group with therelated airway administration were carried out by a two-sided student'st-test using SAS. P < 0.05 and P < 0.01 were considered statisticallysignificant and highly statistically significant, respectively. The datawere presented as the mean ± standard error.

3. Results and discussion

3.1. Physicochemical properties of nanoparticles

We used TiO2, ZnO, and ZrO2 nanoparticles with diameters of100 nm (n100TiO2, n100ZnO, and n100ZrO2), 50 nm (n50TiO2,n50ZnO, and n50ZrO2), and 10 nm (n10TiO2, n10ZnO, and n10ZrO2) tostudy the effect of the particle diameter on the distribution of theparticles in breast milk and their translocation to offspring. SEM con-firmed that all of the nanoparticles were smooth-surfaced spheres andhad the expected size (Supplementary Fig. S2-a). For nanoparticles ofall sizes in water and serum, the size distribution spectra obtained bythe dynamic light scattering analysis showed a larger peak than theexpected size and the size observed in the SEM images (SupplementaryFig. S2-b,c for hydrodynamic diameters, zeta potentials, and pH va-lues). Compared with the nanoparticles in water solution, those inserum had relatively large hydrodynamic diameters (SupplementaryFig. S2-b,c). The larger-than-expected size of the nanoparticles in so-lution (water and serum) may be attributed to the aggregation amongthe nanoparticles (Salameh et al., 2014; Zook et al., 2011). The ob-served particle sizes of the nanomaterials (TiO2, ZrO2, and ZnO of allsizes) in the serum were relatively larger than those in the water. TiO2,ZrO2, and ZnO of the same size had similar specific surface areas. For aspecific nanoparticle, the specific surface area is reduced as the nano-particle size increased (Supplementary Fig. S2-c). As the specific surfacearea of the nanoparticles increased, the toxicity of nanoparticles to-wards the mammalian cells increased (Part et al., 2018; Xiong et al.,2013). In summary, all of the nanoparticles had a similar morphology(smooth-surfaced spheres) and aggregation. The specific surface areacan be related to the particle size rather than the chemical composition.Although aggregation was observed among all of the nanoparticles usedin the current study, the degree of aggregation largely depended on theparticle size (Penn et al., 2007).

3.2. Effects of nanoparticle size on animal performance

The milk yield of dams with oral exposure was not changed by ei-ther the particle size or the chemical composition of the nanomaterials(P > 0.05, Fig. 1-a). The BW change of dams with different treatmentsare presented in Fig. 2-a. Dams with oral n10ZnO exposure had a lowerBW increase than that of the control dams at the first week postpartum(P < 0.05, Fig. 2-a). Dams with oral n10TiO2 exposure had a higherBW increase relative to the dams in the control group in the secondweek postpartum (P < 0.001, Fig. 2-a). In the second week after birth,a lower BW of offspring that consumed milk from dams with oraln10TiO2, n10ZnO and n10ZrO2 exposure (n10TiO2-O-, n10ZnO-O-, andn10ZrO2-O-offspring, respectively) was observed compared with thosein the control group (P < 0.05, Fig. 2-b). In the third week after birth,lower BW were identified in n10TiO2-O– and n10ZnO-O-offspring

relative to those of ones in the control group (P < 0.05, Fig. 2-b).n10TiO2-O-, n10ZnO-O-, and n10ZrO2-O-offspring had lower survivalrates (< 80% 3weeks after birth) than the offspring in the controlgroups and the offspring that consumed milk from dams exposed to 50or 100 nm diameter nanoparticles (Fig. 2-c). To our knowledge, this isthe first study investigating the effects of nanomaterials (types andparticle size) on the milk production of dams. Morishita et al. found thatthe apoptosis rate and tight junction integrity of the mammary glandwere similar in lactating mice with or without nanosilver, but the milkyield data were not available (Morishita et al., 2016). Moreover, the BWchange of dams with nanoparticle exposure (n10ZnO and n10TiO2) aswell as the growth retardation and decreased survival rate of offspringthat consumed their milk suggested that the oral exposure to TiO2,ZrO2, and ZnO, especially when the particle size is 10 nm, was harmfulto both dams and offspring. Our observation is consistent with those ofYang et al., who found that the injection of quantum dots in lactatingrats reduced the weight gain of their neonates (Yang et al., 2018). Insummary, the biological effect of nanoparticles (TiO2, ZrO2, and ZnO)on the health of dams and neonates depends on the particle size(Behzadi et al., 2017; Hadjidemetriou and Kostarelos, 2017; Satzeret al., 2016; Talamini et al., 2017). Exposure to TiO2, ZrO2, and ZnOwith 10 nm diameters had the most significant effect on the BW changesof dams and the BW loss of offspring that consumed their milk. Thus,TiO2, ZrO2, and ZnO nanoparticles with 10 nm diameters were selectedfor use in the next experiment.

3.3. Transfer of nanoparticles into breast milk

When dams consumed n10TiO2, n10ZnO and n10ZrO2 via the oral/airway routes (n10TiO2-O-, n10ZnO-O-, and n10ZrO2-O-dams;n10TiO2-A-, n10ZnO-A- and n10ZrO2-A-dams), the concentrations ofTi, Zn and Zr in the blood were higher than in dams without nano-particle exposure (Fig. 3-a). The Ti and Zn concentrations in bloodwithin 2 h after exposure were higher in n10TiO2-A-dams and n10ZnO-A-dams compared with those in n10TiO2-O– and n10ZnO-O-dams, re-spectively (Fig. 3-a). However, n10ZrO2-A-dams had relatively lower Zrconcentrations compared to the n10ZrO2-O-dams within the first 12 hpost-nanoparticle exposure (Fig. 3-a). In terms of the dynamic changesin the nanoparticles in milk, all dams with nanoparticle exposure hadhigher concentrations of Ti, Zn or Zr in their milk throughout the 12 hafter nanoparticle exposure compared to animals without nanoparticleexposure (Fig. 3-b). Throughout the 12 h post-exposure period,n10TiO2-A- and n10ZrO2-A-dams had higher milk nanoparticle con-centrations compared with those of n10TiO2-O– and n10ZrO2-O-dams(Fig. 3-b). However, the n10ZnO-A-dams had higher milk Zn con-centrations than the n10ZnO-O-dams only within 2 h post-administra-tion (Fig. 3-b). In terms of the nanoparticle clearance efficiency inmaternal blood, n10TiO2-O-, n10ZnO-O-, and n10ZrO2-O-dams hadhigh, medium and low elimination rate constants as well as low,medium and high half-lives, respectively (Supplementary Fig. S3-b, d,f). These findings indicated that exposure by the airway route had in-creased the appearance of the nanoparticles in blood and their trans-location in milk in dams compared to those exposed orally, regardlessof the nanomaterial category. These observations can be attributed tothe surface coating of organics/proteins in the gastrointestinal tract thatprotects from oral nanoparticle exposure (Ngamchuea et al., 2018;Sohal et al., 2018). However, the efficiency of nanoparticle transfer intomilk varied for different nanomaterials (Zn > Zr > Ti), as indicatedby the milk/blood ratio (calculated by the time-course AUC for milk

Fig. 4. Distributions of nanoparticles within different components of breast milk. The Ti (a), Zn (b) and Zr (c) concentrations or content in the skim milk, milksomatic cells, and each milk cell type were determined on experiment day 12 (lactating day 21 for dams). The ratio of nanoparticles in skim milk to whole milk wasalso calculated. Ti and Zr in milk somatic cells and each milk cell type of control groups was not detected (ND) due to their low mass which was lower than the limitof detection for inductively coupled plasma mass spectrometry. -A represents airway exposure, eO represents oral exposure. The data are presented as themean ± standard error (n=9). * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001.


9

Fig. 5. Biodistributions of Ti, Zn and Zr in maternal and offspring mice after nanoparticle exposure to maternal mice for 21 days. The bio-distributions of Ti, Zn andZr in the breast, liver, spleen, lungs, kidneys, intestine and brain of maternal (a) and offspring (b) mice were measured on experiment day 21 (lactating day 21 fordams and postnatal day 21 for offspring). -A represents airway exposure, eO represents oral exposure. The data are presented as the mean ± standard error (n=6).* represents the significance between the oral/airway nanoparticle exposure group and control. # represents the significance between the oral nanoparticle exposuregroup and airway nanoparticle exposure group. * or # represents P < 0.05, ** or ## represents P < 0.01, *** or ### represents P < 0.001, and **** or ####represents P < 0.0001.


10



11

divided by the AUC for blood) (Fleishaker, 2003). This finding wasconsistent with findings on lactating mice with exposure to other metalnanomaterials (Ag and Au), and the varied transfer ability could beattributed to the chemical and surface characteristics of the differenttypes of nanomaterials (Morishita et al., 2016).

The distribution of the nanoparticles in the different components ofmilk was further determined (Fig. 4). Compared with control dams, alldams with nanoparticle exposure had increased Ti, Zn or Zr con-centrations in milk SCs and skim milk. However, n10TiO2-O– andn10ZnO-O-dams had greater ratios of Ti or Zn in skim milk than inwhole milk when compared with those of n10TiO2-A- and n10ZnO-A-dams. The mammary gland is an important system that is responsiblefor translocating substances between the mother and offspring; for thisreason, lactating mothers need to be cautious when consuming foods ordrugs, as they might contain substances that are potentially toxic tooffspring (Sachs, 2013). Based on our data, the translocation of allnanoparticles from the blood to the milk depended on two pathways:the mammary epithelial cell-dependent pathway (indicated by themetal concentrations in skim milk and within the mammary epithelialcell, and ratio of the metal concentration in skim milk to whole milk)and the immune cell-dependent pathway (indicated by the metal con-centration in each milk immune cell). In this study, we observed thatthe nanoparticle transfer from the blood to the milk differed with theexposure pathway. For all nanoparticle exposures, the immune cell-dependent pathway seemed to play a more important role in the airwayexposure route than the oral exposure route, as the absolute amountand percentage of exposed metal was higher in the milk NEU for theairway exposure route than for the oral exposure route. The percentageof metal in the NEU accounted for> 65% of metal in the whole milk SCin all airway exposures to the nanoparticles, whereas the percentage ofmetal in the NEU accounted for< 30% in the oral exposure route.These findings may suggest that a strong intestinal barrier was formed(digestive fluids and enzymes, mucus, microbes, and mutilayers of in-testinal cells with tight junctions) to prevent the blood from beingcontaminated by the ingestion of n10TiO2 and n10ZnO. As the barrierin the lungs (the thin pulmonary alveoli) is weaker, all dams withairway exposure finally compensated by changes of immune cells(Sinnecker et al., 2014; Sung et al., 2007). This is consistent with therelatively greater immune response in dams with airway exposure(higher %NEU in the blood and milk, Supplementary Fig. S4 and Fig. 1-c). Thus, the utilization of nanoparticles should be reconsidered due totheir potential safety issue, especially in breastfeeding mothers andtheir offspring.

The nanoparticle distributions on ED 21 in important tissues arepresented in Fig. 5-a. Compared with those of the control dams,n10TiO2-O-dams had higher Ti concentrations in the breast, liver, andbrain, and n10TiO2-A-dams had higher Ti concentrations in the breast,liver, spleen, lungs, kidneys and brain. Moreover, n10TiO2-A-dams hadhigher Ti levels in the spleen, lungs, kidneys and brain tissues comparedwith n10TiO2-O-dams. Compared with dams in the control group, then10ZnO-O-dams had higher Zn concentrations in the breast, liver,spleen and kidneys, and the n10ZnO-A-dams had higher Zn con-centrations in the breast, liver, spleen, lungs, kidneys and brain. Inaddition, higher Zn levels in the liver, spleen, lungs, kidneys and brainwere observed in the n10ZnO-A-dams compared to those of then10ZnO-O-dams. Finally, compared with the control-dams, the

n10ZrO2-O-dams had increased Zr concentrations in the breast, spleen,kidneys and intestine, whereas n10ZrO2-A-dams had higher Zr con-centrations in the breast, liver, spleen, lungs, kidneys and brain. Similarto ZnO exposure, n10ZrO2-A-dams had higher Zr concentrations in thebreast, liver, spleen, and brain but a lower Zr content in the small in-testine compared with the n10ZrO2-O-dams. These results indicated theextensive accumulation of the metal element of the exposed nano-particles in more than one important tissue regardless of the exposureroute. However, the nanoparticles accumulation seemed to be differentin some tissues of dams with different exposure routes. For example, thegreater amount of nanoparticles in n10ZnO-A- and n10TiO2-A-damsrelative to dams that were exposed via the oral route suggested that theinhaled exposure to nanoparticles could be of higher risk than the oralexposure in lactating animals, which is in agreement with a previousstudy (Hougaard et al., 2010). The airway exposed particles woulddeposit in the lungs and accumulate with every additional exposure, incontrast to the gavaged particles, and translocate through the lungepithelium to other organs, although the rate of distribution varies(Hougaard et al., 2010; Rossi et al., 2010). In addition, TEM analysiswas performed on the mammary glands of dams to confirm that thenanoparticles were at least partly delivered in the nanoparticle form,not in their ionic form (Supplementary Fig. S5). For all of the nano-particle treatments, the nanoparticles were present in the mammarygland, indicating their ability to translocate from the gastrointestinal orrespiratory tracts to the mammary gland, and further transfer to off-spring.

Fig. 6-a and -b present the effects of the oral and airway exposureroutes of nanoparticles, respectively, on the tight junction and apop-tosis status in the mammary gland of dams. The mammary glands ofn10TiO2-O-, n10ZnO-O– and n10ZrO2-O-dams had reduced the claudin-3 staining intensity compared with those of the control dams (Fig. 6-a)and consistently had a higher apoptosis rate. Moreover, Zhang et al.found that TiO2 nanoparticles caused a disrupted tight junction of theblood-milk barrier as indicated by the loss of tight junction proteins(Zhang et al., 2015). During airway exposure, only the n10ZnO-A-damshad lower claudin-3 intensity and higher apoptosis rate in the mam-mary gland relative to the control-dams, which suggested that the up-take of n10ZnO by the immune cells was not sufficient to protect themammary gland. In addition, the n10ZrO2-O-dams had a lower stainingdensity in the mammary gland than n10ZrO2-A-dams. These resultsshowed that the mammary gland structure can be impaired to differentdegrees by the nanoparticle exposure depending on the category of thenanoparticle and the exposure route. All of the nanoparticle exposuresinduced high apoptosis rates for the mammary gland (Fig. 6-b), sug-gesting they play harmful roles in the mammary gland. These resultswere consistent with the cytotoxicity results of nanoparticles for humanbreast cells and their mammary tumorigenesis traits (Peng et al., 2019;White et al., 2019).

In terms of milk production, the milk yield was reduced in then10ZnO-A-dams compared to the control dams (P < 0.05, Fig. 1-a),whereas other treatments did not alter the milk yield of dams. In termsof milk composition, the n10ZrO2-O– and n10ZnO-O-dams had lowermilk fat concentrations than the control-dams (P < 0.05, Fig. 1-b). TheROS concentrations in the milk of n10TiO2-O– and n10ZnO-A-damswere higher than in those of the control animals (P < 0.05). However,the concentrations of albumin, IgA, lactose, protein, SC count, and β-

Fig. 6. The mammary gland structure and mammary epithelial cell viability of the lactating dams exposed to nanoparticles in oral or airway route on experiment day12 (lactating day 12). (a) Immunohistochemistry with the anti-Claudin-3 antibody showed the integrity of the tight junctions between mammary epithelial cells toindicate whether the mammary gland structure was destroyed by nanoparticles (Magnification: 400×). Claudin-3 was stained in orange; the nucleus was stained inblue. (b) Immunofluorescence TUNEL apoptosis analysis showed the cell apoptosis conditions to indicate how the mammary epithelial cell viability was affected bynanoparticles (Magnification: 200×). TUNEL-positive cells were stained in green; the nucleus was stained in blue. (c) The corrected staining intensity of Claudin-3was calculated by using 6 visual fields around the parts of mammary acini of each slice under a microscope at a magnification of 400×. (d) The apoptotic index ofeach slice was calculated by the percentage of TUNEL-positive nuclei relative to the total number of nuclei under a microscope at a magnification of 200×. The datafor (c) and (d) are presented as the mean ± standard error (n=3). * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. (For inter-pretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


12



13

casein in the blood were not affected by nanoparticle exposure(P > 0.05).

The lower milk fat concentration may be attributed to the lower fatbiosynthesis in dams induced by the consumption of Zn and Zr. Relativeto dams exposed to nanoparticles through the airway routes, damsexposed to nanoparticles through the oral route showed a lower fatbiosynthesis rate, possibly due to a dysfunction of gastrointestinal mi-crobes, which greatly contribute to the supply of milk fat precursors(Bergin and Witzmann, 2013). Finally, exposure to n10ZnO andn10TiO2 may lead to increased oxidative stress in mice (Shrivastavaet al., 2014), which further increased the ROS accumulation in the milk.

3.4. Distribution of nanoparticles to offspring via breast milk

We further determined the nanoparticle distribution in the blood ofoffspring after they consumed milk from their mothers with/withoutnanoparticle exposure (Supplementary Fig. S6). We found that the na-noparticle concentrations in the blood were higher in all offspring ofdams with nanoparticle exposure compared with those in the controloffspring (P < 0.05, Supplementary Fig. S6). Moreover, the bloodmetal concentrations were higher in the offspring of dams with airwaynanoparticle exposure (n10TiO2-A-, n10ZnO-A-, and n10ZrO2-A-off-spring) compared to the offspring of dams with oral nanoparticle ex-posure (P < 0.05, Supplementary Fig. S6). In terms of the nanoparticleclearance efficiency in the blood of offspring, n10ZnO-O– and n10ZrO2-O-offspring had relatively lower elimination rate constants as well asrelatively higher half-lives compared with those of n10TiO2-O-offspring(Supplementary Fig. S3-b, d, f). However, the metal type did not affectthe clearance efficiency and half-life of offspring whether they con-sumed milk from dams with either oral or airway exposure routes.

In terms of the metal distribution in tissues, the Ti concentrations inthe liver, spleen, kidneys and brain were higher in n10TiO2-O– andn10TiO2-A-offspring than in control offspring (P < 0.05, Fig. 5-b). Inaddition, the Ti concentrations in the spleen and kidneys were higher inn10TiO2-A-offspring than in control- and n10TiO2-O-offspring(P < 0.05, Fig. 5-b). n10ZnO-A-offspring had higher Zn concentrationsin the liver, spleen, lungs, kidneys, intestine and brain compared withcontrol offspring (P < 0.05, Fig. 5-b). n10ZnO-A-offspring also hadhigher Zn concentrations in the liver, spleen, lungs and kidneys com-pared to those of n10ZnO-O-offspring (P < 0.05, Fig. 5-b). Similarly,n10ZrO2-A-offspring had higher Zr concentrations in the liver, spleen,lungs, kidneys and brain compared with control offspring (P < 0.05,Fig. 5-b). Additionally, n10ZrO2-A-offspring had higher Zr concentra-tions in the spleen and lungs compared with those of n10ZrO2-O-off-spring and control offspring (P < 0.05, Fig. 5-b). Our results are con-sistent with quantum dot accumulation profiling in rat offspringthrough milk transfer (Yang et al., 2018), indicating that maternalnanoparticle exposure, either in an oral or airway route, will be de-posited in important tissues of offspring after breastfeeding.

3.5. Biological effects of nanoparticles on offspring exposed via breast milk

The BW of offspring from the nanoparticle-exposed dams was sig-nificantly reduced compared with that of the control animals(P < 0.05, Fig. 7-a). Although the milk production of dams with na-noparticle exposure was not affected, with the exception of the

n10ZnO-A-dams, the nanoparticles entering into the milk of damsthrough oral/airway exposure play an important role in inhibiting thegrowth rate of offspring consuming the milk. Breastfeeding was re-commended globally, as it provides nutrients and immunity to offspring(Victora et al., 2016). However, offspring that were weaned early had agreater BW than offspring that were not if they consumed contaminatedmilk (P < 0.05, Fig. 7-b), indicating that nanoparticles in the milk,rather than compositional change, inhibited the growth of offspring.Moreover, offspring from both airway/oral nanoparticle-exposed damshad lower survival rates 6 weeks after birth than those of the control(P < 0.05, Fig. 7-c). Thus, the relatively lower survival rate of thisstudy is associated with the lower BW in offspring consuming nano-particle-contaminated milk.

The results of the hematology analysis for offspring are presented inSupplementary Fig. S7. n10TiO2-O-offspring had higher %MONO andPDW in the blood than the control offspring (P < 0.05, SupplementaryFig. S7). The n10ZnO-O-offspring had lower RBC than the control off-spring (P < 0.05, Supplementary Fig. S7). In terms of airway exposure,increased blood cell concentrations were observed in n10TiO2-A-off-spring (LYM and NEU), n10ZnO-A-offspring (NEU, HGB and WBC), andn10ZrO2-A-offspring (NEU), compared with those corresponding bloodcell concentrations of the control-offspring (P < 0.05, SupplementaryFig. S7). In terms of the different exposure routes, n10TiO2-A-offspringhad higher blood %RETIC than n10TiO2-O-offspring (P < 0.05,Supplementary Fig. S7). n10ZnO-A-offspring had a higher HGB andRBC in the blood than the n10ZnO-O-offspring (P < 0.05,Supplementary Fig. S7).

The systematic physiological immune response is a key factor inregulating the growth efficiency of infants (Simon et al., 2015). Theincreased NEU and decreased LYM in the blood of n10TiO2-A-offspringsuggested that milk contaminated with nanoparticles via the airwayroute led to systematical oxidative stress in the mouse model (Shahinet al., 2013). By contrast, the increased blood PDW in n10TiO2-O-off-spring indicated a blood immune cell disorder (Li et al., 2017). Theresults indicated that milk from n10TiO2-O– and n10TiO2-A-dams ledto the growth inhibition of the breastfeeding offspring via differentroutes. A previous clinical study indicated that infants consuming ametal-fortified formula had greater increases in blood HGB (Moffattet al., 1994). Our study revealed that compared with the n10ZnO-O-offspring, the n10ZnO-A-offspring had a weaker state of health. Thehematology variables in the n10ZnO-O-offspring were similar to thoseof the control offspring, suggesting that the n10ZnO-O-dams did notimpact the health status and functionality of the blood immune cells inoffspring consuming their milk. However, consistent with changes inrats and human exposed to nanoparticles, the increased HGB, NEU andWBC in the n10ZnO-A-offspring indicated that the ZnO airway exposurein dams led to an inflammatory reaction in offspring (Aztatzi-Aguilaret al., 2016; Braakhuis et al., 2014; Monsé et al., 2018). This result isconsistent with the higher Zn concentration contamination in the smallintestine of the n10ZnO-A-offspring and its pathological change. Ac-cording to the results from human and monkey kidney cell lines, theincreased blood NEU in n10ZrO2-A-offspring suggested that milk fromn10ZrO2-A-dams led to the reduced BW of these offspring by increasingthe immune response (Keceli and Alanyali, 2004; Simon et al., 2015).Moreover, just as the nanoparticle-induced immune response increasedthe circulation of NEU in human blood (Monsé et al., 2018), the higher

Fig. 7. Body weight and survival rate of offspring mice fed milk from maternal mice with nanoparticle exposure. (a) Body weight of offspring mice fed milk frommaternal mice with nanoparticle exposure in oral or airway route. (b) Body weight of offspring mice with/without early weaning fed milk from maternal mice withnanoparticle exposure in oral or airway route. Early weaning of neonates was performed in the group in which lactating dams were exposed to nanoparticles at week2 after birth. (c) Survival rate of offspring mice fed milk from maternal mice with nanoparticle exposure in oral or airway route. -A represents airway exposure, eOrepresents oral exposure, and -E represents early weaning. The data are presented as the mean ± standard error for (a) and (b) (n=9 for weeks 0 and 1 after birth,and n= 6 for week 2 to week 6 after birth) and are presented as a single calculation value for (c). * represents the significance between the oral/airway nanoparticleexposure group and control group. # represents the significance between the oral/airway nanoparticle exposure group with early weaning and control group. * or #represents P < 0.05, ** or ## represents P < 0.01, *** or ### represents P < 0.001, and **** or #### represents P < 0.0001.


14

Fig. 8. The pathological analysis of the main organsin the neonates breastfed by maternal mice exposedto 10 nm nanoparticles using HE staining on experi-ment day 21. (a) Liver pathological analysis. A blackarrow represents edema or ballooning degeneration;a red arrow represents hepatic sinus congestion. (b)Spleen pathological analysis. A black arrow re-presents reduced white pulp; a red arrow representsextramedullary hematopoietic cell aggregation; ayellow arrow represents granulocytic infiltration;and a green arrow represents the irregular mor-phology of white pulp. (c) Lung pathological ana-lysis. A black arrow represents the massive thick-ening of the alveolar walls; a red arrow representslessened or expanded alveolar lumen; a yellow arrowrepresents inflammatory cell infiltration; a greenarrow represents capillary congestion; and a bluearrow represents alveolar stenosis. (d) Kidney pa-thological analysis. A black arrow represents edemadegeneration; a red arrow represents renal tubularinterstitial congestion; and a yellow arrow representsthe existence of eosinophilic proteinoid and the for-mation of renal cast. (e) Intestine pathological ana-lysis. A black arrow represents edema of the laminapropria of intestinal villi and the interval broadeningbetween the lamina propria and epithelium. (f) Brainpathological analysis. A black arrow represents ery-throcyte aggregation; a red arrow represents thecontraction and hyperchromation of neurons(n=3). (For interpretation of the references to colorin this figure legend, the reader is referred to the webversion of this article.)


15

blood NEU in offspring consuming milk from airway-exposed dams maybe attributed to the higher nanoparticle concentrations in their bloodrelative to that of oral-exposed dams.

Although dams with nanomaterial exposure through the oral routeseemed to have a less negative effect on the hematological profiles oftheir offspring relative to those with airway nanoparticle exposure,pathological changes were observed in the main tissues in all offspringthat consumed milk from dams with nanoparticle exposure. Thesechanges included liver edema/ballooning degeneration (in n10TiO2-A-,n10ZnO-A-, n10ZrO2-A-, and n10TiO2-O-offspring) and hepatic sinuscongestion (in the n10ZnO-A-offspring). In the spleen, varied patholo-gical changes were observed (in n10TiO2-A-, n10ZnO-A-, n10ZrO2-A-,n10TiO2-O-, and n10ZrO2-O-offspring). In the lung tissue, massivethickening of the alveolar walls, altered size of the alveolar lumen andinflammatory cell infiltration were observed in the offspring of damswith airway nanoparticle exposure. In addition, lung capillary conges-tion (in n10ZrO2-A- and n10ZrO2-A-offspring) and alveolar stenosis (inn10TiO2-O– and n10ZrO2-O-offspring) were identified. For all offspringthat consumed milk from dams with nanoparticle exposure, edemadegeneration was observed in the kidneys. Additionally, renal tubularinterstitial congestion (in n10TiO2-O-, n10ZrO2-O– and n10ZrO2-A-offspring) and eosinophilic proteinoid/renal cast formation (inn10ZnO-O-offspring) were observed. In the intestinal region, n10ZnO-A-offspring had lamina propria edema of the intestinal villi. Finally,erythrocyte aggregation (in n10TiO2-O– and n10TiO2-A-offspring) andthe contraction and hyperchromation of neurons (in the n10ZnO-A- andn10ZrO2-A-offspring) were observed in the brain.

The histology and function of organs can be changed by nano-particle accumulation in the gills, liver, heart and brain of zebrafish(Chen et al., 2011). Compared with animals in other groups, then10ZnO-O-offspring only had pathological changes in the kidney region(Fig. 8-d). This observation is consistent with the increased Zn con-centration in the kidneys of the n10ZnO-O-offspring. Similarly, limitedmorphological changes in the small intestine of n10TiO2-A- andn10ZrO2-A-offspring and offspring of dams with oral nanoparticle ex-posure may contribute to the similar concentrations of nanoparticles inthe small intestine in these offspring relative to the control offspring.Moreover, a previous study in mice indicated that, due to differenttoxicological traits, the oral exposure to different nanoparticles (TiO2

and ZnO) induced tissue injuries after uptake by the gastrointestinaltract via different mechanisms of action (Shrivastava et al., 2014). Inthis study, the histological changes observed in offspring consumingmilk from dams with varied nanoparticle exposure (TiO2, ZnO andZrO2), such as in the spleen and lungs, suggested that the change in thehistological function of tissues in infants exposed to nanomaterials viabreastfeeding had different toxicological traits. Moreover, offspring thatconsumed milk from dams with different exposure routes had differenthistological changes. For example, n10ZnO-A-offspring had significantmorphological changes in the liver, spleen, lungs, intestine and braincompared with n10ZnO-O-offspring, while n10ZrO2-O– and n10ZrO2-A-offspring had different histological changes in the spleen, liver andbrain. The evidence suggested that when dams were exposed to nano-particles via different pathways, the potential toxicological impacts onoffspring were different from each other. The mammary gland is a keysystem in processing and delivering nutrients from the mother to heroffspring (Fleishaker, 2003; McManaman and Neville, 2003; Sachs,2013). Maternal exposure to nanoparticles via the airway route seemedto rely more on the immune cell-dependent pathway than the oralroute. Thus, the different histological changes in the tissues of offspringthat consumed contaminated milk from dams with different exposureroutes could be related to the different biological distributions of thenanoparticles in the milk. Based on the nanoparticle concentrations andmorphological changes of offspring, it is acknowledged that motherswith airway exposure to nanomaterials had a relatively greater tox-icological effect on their breastfeeding offspring compared to dams withoral exposure.

4. Conclusions

This study demonstrated that both the oral and airway exposure tonanoparticles by lactating dams led to changes in their BW as well aslower BW and survival of their breastfeeding offspring through a toxi-city mechanism that depends on the particle size, chemical type, andexposure route. The BW, systematic physiological immune response andmilk production (milk yield and composition) can all be changed bynanoparticles in chemical-dependent (n10TiO2, n10ZnO, and n10ZrO2)and exposure duration-dependent (oral and airway) manners due totheir different invasion manners, tissue distribution profiles and milktransfer pathways. More importantly, when milk containing nano-particles via either oral or airway exposure of dams was consumed bytheir offspring, a reduced BW as well as systematic dysfunction (inblood cells and main tissues) were observed, which was attributed tothe transfer of nanoparticles into the blood and tissues. Our resultsindicated that maternal nanoparticle exposure via the airway routecould be more severe than that of the oral route in terms of the BW andsurvival rate of the infants. The study proposed a new toxicologicalassessment of nanoparticle exposure during lactation by consideringboth the mother and offspring. Our study indicated that nanomaterials(TiO2, ZnO, and ZrO2, present in household products, cosmetics andpersonal care products), which are increasingly accessible to breast-feeding women, need to be reconsidered to keep breastfeeding safe.Moreover, it is urgent to limit the application of potentially toxic na-noparticles, which can be translocated to the breast milk and subse-quently to offspring, to provide offspring with a healthy growing en-vironment.

Declaration of competing interest

The authors declare that they have no known competing financialinterests or personal relationships that could have appeared to influ-ence the work reported in this paper.

Acknowledgment

The authors appreciate the staff of Faculty of Agricultural, Life, andEnvironmental Sciences of Zhejiang University for their assistant onphysicochemical characterization of nanoparticles.

Funding

This study was supported by Natural Science Foundation of China(grant numbers: 31930107), Natural Science Foundation of China(grant numbers: 31872380), Fundamental Research Funds for theCentral Universities (grant numbers: 2-2050205-18-237) andAgriculture Research System of China (grant numbers: CARS37).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.envint.2019.105153.

References

Anklam, E., et al., 2011. Impact of Engineered Nanomaterials on Health: Considerationsfor Benefit-Risk Assessment. Publications Office of the European Union, Luxembourg.

Aztatzi-Aguilar, O.G., et al., 2016. Early kidney damage induced by subchronic exposureto PM2.5 in rats. Part. Fibre Toxicol. 13, 68.

Behzadi, S., et al., 2017. Cellular uptake of nanoparticles: journey inside the cell. Chem.Soc. Rev. 46, 4218–4244.

Bergin, I.L., Witzmann, F.A., 2013. Nanoparticle toxicity by the gastrointestinal route:evidence and knowledge gaps. Int. J. Biomed. Nanosci. Nanotechnol. 3 (1–2).

Black, R.E., et al., 2013. Maternal and child nutrition study group. Maternal and childundernutrition and overweight in low-income and middle-income countries. Lancet382, 427–451.

Braakhuis, H.M., et al., 2014. Particle size dependent deposition and pulmonary


16



http://refhub.elsevier.com/S0160-4120(19)31927-0/rf0005












inflammation after short-term inhalation of silver nanoparticles. Part. Fibre Toxicol.11, 49.

Braun, D., et al., 2018. Monitoring early life mycotoxin exposures via LC-MS/MS breastmilk analysis. Anal. Chem. 90, 14569–14577.

Čechová, E., et al., 2017. Legacy and alternative halogenated flame retardants in humanmilk in Europe: implications for children's health. Environ. Int. 108, 137–145.

Chen, J., et al., 2011. Effects of titanium dioxide nano-particles on growth and somehistological parameters of zebrafish (danio Rerio) after a long-term exposure. Aquat.Toxicol. 101, 493–499.

Deepthi, S., et al., 2016. An overview of chitin or chitosan/nano ceramic compositescaffolds for bone tissue engineering. Int. J. Biol. Macromol. 93 (Pt B), 1338–1353.

Ema, M., et al., 2016. Reproductive and developmental toxicity of carbon-based nano-materials: a literature review. Nanotoxicology 10, 391–412.

Ema, M., et al., 2017. A review of reproductive and developmental toxicity of silvernanoparticles in laboratory animals. Reprod. Toxicol. 67, 149–164.

Fleishaker, J.C., 2003. Models and methods for predicting drug transfer into human milk.Adv. Drug Deliv. Rev. 55, 643–652.

Hadjidemetriou, M., Kostarelos, K., 2017. Nanomedicine: evolution of the nanoparticlecorona. Nat. Nanotech. 12, 288–290.

Hao, Y., et al., 2017. Molecular evidence of offspring liver dysfunction after maternalexposure to zinc oxide nanoparticles. Toxicol. Appl. Pharmacol. 329, 318–325.

Hassiotou, F., et al., 2013. Maternal and infant infections stimulate a rapid leukocyteresponse in breastmilk. Clin. Transl. Immunol. 2, e3.

Hougaard, K.S., et al., 2010. Effects of prenatal exposure to surface-coated nanosizedtitanium dioxide (UV-titan). A study in mice. Part Fibre Toxicol 7, 16.

Hougaard, K.S., et al., 2015. A perspective on the developmental toxicity of inhaled na-noparticles. Reprod. Toxicol. 56, 118–140.

Illuminato, I., Friends of the Earth U.S, 2016. Nanoparticles in Baby Formula: Tiny NewIngredients Are a Big Concern. pp. 1–31.

Jones, G., et al., 2003. How many child deaths can we prevent this year? Lancet 362,65–71.

Kassandra, W., et al., 2014. Milk collection methods for mice and Reeves' muntjac deer. J.Vis. Exp.(89), e51007.

Keceli, S., Alanyali, H., 2004. A study on the evaluation of the cytotoxicity of Al2O3,Nb2O5, Ta2O5, TiO2, and ZrO2. Turk. J. Eng. Environ. Sci. 28, 49–54.

Landrigan, P.J., et al., 2002. Chemical contaminants in breast milk and their impacts onchildren’s health: an overview. Environ. Health Perspect. 110, A313–A315.

Lehmann, G.M., et al., 2014. Improving the risk assessment of lipophilic persistent en-vironmental chemicals in breast milk. Crit. Rev. Toxicol. 44, 600–617.

Li, Z., et al., 2017. Preoperative red cell distribution width and neutrophil-to-lymphocyteratio predict survival in patients with epithelial ovarian cancer. Sci. Rep. 7, 43001.

McManaman, J.L., Neville, M.C., 2003. Mammary physiology and milk secretion. Adv.Drug Deliv. Rev. 55, 629–641.

Melnik, E.A., et al., 2013. Transfer of silver nanoparticles through the placenta and breastmilk during in vivo experiments on rats. Acta Nat. 5, 107–115.

Moffatt, M.E., et al., 1994. Prevention of iron deficiency and psychomotor decline in high-risk infants through use of iron-fortified infant formula: a randomized clinical trial. J.Pediatr. 125, 527–534.

Mohammadipour, A., et al., 2013. The effects of exposure to titanium dioxide nano-particles during lactation period on learning and memory of rat offspring. Toxicol.Ind. Health 32, 221–228.

Monsé, C., et al., 2018. Concentration-dependent systemic response after inhalation ofnano-sized zinc oxide particles in human volunteers. Part. Fibre Toxicol. 15, 8.

Morishita, Y., et al., 2016. Distribution of silver nanoparticles to breast milk and theirbiological effects on breast-fed offspring mice. ACS Nano 10, 8180–8191.

Nabhan, A.F., 2015. WHO recommendations on interventions to improve preterm birthoutcomes. In: WHO Guidelines Approved by the Guidelines Review Committee.World Health Organization, Geneva, Switzerland, pp. 92.

Ngamchuea, K., et al., 2018. The fate of silver nanoparticles in authentic human saliva.Nanotoxicology 12, 305–311.

Ou, L., et al., 2018. Physiologically based pharmacokinetic (PBPK) modeling of humanlactational transfer of methylmercury in China. Environ. Int. 115, 180–187.

Part, F., et al., 2018. A review of the fate of engineered nanomaterials in municipal solid

waste streams. Waste Manag. 75, 427–449.Peng, F., et al., 2019. Nanoparticles promote in vivo breast cancer cell intravasation and

extravasation by inducing endothelial leakiness. Nat. Nanotechnol. 14, 279–286.Penn, R.L., et al., 2007. Size dependent kinetics of oriented aggregation. J. Cryst. Growth

309, 97–102.Rossi, E.M., et al., 2010. Airway exposure to silica-coated TiO2 nanoparticles induces

pulmonary neutrophilia in mice. Toxicol. Sci. 113, 422–433.Sachs, H.C., 2013. The transfer of drugs and therapeutics into human breast milk: an

update on selected topics. Pediatrics 132, e796–e809.Salameh, S., et al., 2014. Contact behavior of size fractionated TiO2 nanoparticle ag-

glomerates and aggregates. Powder Technol. 256, 345–351.Sampson, D.A., Jansen, G.R., 1984. Measurement of milk yield in the lactating rat from

pup weight and weight gain. J. Pediatr. Gastr. Nutr. 3, 613.Satzer, P., et al., 2016. Protein adsorption onto nanoparticles induces conformational

changes: particle size dependency, kinetics, and mechanisms. Eng. Life Sci. 16,238–246.

Shahin, S., et al., 2013. 2.45 GHz microwave irradiation-induced oxidative stress affectsimplantation or pregnancy in mice, mus musculus. Appl. Biochem. Biotechnol. 169,1727–1751.

Sharma, B.M., et al., 2019. An overview of worldwide and regional time trends in totalmercury levels in human blood and breast milk from 1966 to 2015 and their asso-ciations with health effects. Environ. Int. 125, 300–319.

Shimizu, M., et al., 2009. Maternal exposure to nanoparticulate titanium dioxide duringthe prenatal period alters gene expression related to brain development in the mouse.Part. Fibre Toxicol. 6, 20.

Shrivastava, R., et al., 2014. Effects of sub-acute exposure to TiO2, ZnO and Al2O3 na-noparticles on oxidative stress and histological changes in mouse liver and brain.Drug Chem. Toxicol. (3), 336–347.

Simon, A.K., et al., 2015. Evolution of the immune system in humans from infancy to oldage. Proc. Biol. Sci. 282, 20143085.

Sinnecker, H., et al., 2014. The gut wall provides an effective barrier against nanoparticleuptake. Beilstein J. Nanotech. 5, 2092–2101.

Sohal, I.S., et al., 2018. Dissolution behavior and biodurability of ingested engineerednanomaterials in the gastrointestinal environment. ACS Nano 12, 8115–8128.

Stark, W.J., et al., 2015. Industrial applications of nanoparticles. Chem. Soc. Rev. 44,5793–5805.

Stewart, C.P., et al., 2013. Contextualising complementary feeding in a broader frame-work for stunting prevention. Matern. Child Nutr. 9, 27–45.

Sumner, S.C., et al., 2010. Distribution of carbon-14 labeled C60 ([14C]C60) in thepregnant and in the lactating dam and the effect of C60 exposure on the biochemicalprofile of urine. J. Appl. Toxicol. 30, 354–360.

Sung, J.C., et al., 2007. Nanoparticles for drug delivery to the lungs. Trends Biotechnol.25, 563–570.

Talamini, L., et al., 2017. Influence of size and shape on the anatomical distribution ofendotoxin-free gold nanoparticles. ACS Nano 11, 5519–5529.

Tao, F., et al., 2017. Emerging and legacy flame retardants in UK human milk and foodsuggest slow response to restrictions on use of PBDEs and HBCDD. Environ. Int. 105,95–104.

UNICEF, WHO, World Bank, 2016. Joint Child Malnutrition Estimates - Levels and Trends(2016 Edition). http://www.who.int/nutgrowthdb/estimates2015/en/, Accesseddate: 27 September 2016.

Victora, C.G., et al., 2016. Breastfeeding in the 21st century: epidemiology, mechanisms,and lifelong effect. Lancet 387, 475–490.

White, A.J., et al., 2019. Metallic air pollutants and breast cancer risk in a nationwidecohort study. Epidemiology 30, 20–28.

Xiong, S., et al., 2013. Specific surface area of titanium dioxide (TiO2) particles influencescyto- and photo-toxicity. Toxicology 304, 132–140.

Yang, L., et al., 2018. Quantum dots cause acute systemic toxicity in lactating rats andgrowth restriction of offspring. Nanoscale 10, 11564–11577.

Zhang, C., et al., 2015. Induction of size-dependent breakdown of blood-milk barrier inlactating mice by TiO2 nanoparticles. PLoS One 10, e0122591.

Zook, J.M., et al., 2011. Stable nanoparticle aggregates/agglomerates of different sizesand the effect of their size on hemolytic cytotoxicity. Nanotoxicology 5, 517–530.


17
















































































































http://www.who.int/nutgrowthdb/estimates2015/en/













translocation of transition metal oxide nanoparticles to breast...

Documents