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Journal of Health Science, 55(1) 95–102 (2009)

Nanoparticles Transferred from Pregnant Mice to
Their Offspring Can Damage the Genital and Cranial
Nerve Systems
Ken Takeda,∗, a Ken-ichiro Suzuki, b Aki Ishihara, a Miyoko Kubo-Irie, a Rie Fujimoto, a
Masako Tabata, a Shigeru Oshio, c Yoshimasa Nihei, b Tomomi Ihara, dand Masao Sugamata daDepartment of Hygiene Chemistry, Faculty of Pharmaceutical Science, b Department of Pure and Applied Chemistry, Faculty of Scienceand Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278–8510, Japan, c Department of Hygiene Chemistry,
Ohu University School of Pharmaceutical Sciences, 31–1 Misumidou, Tomita-machi, Kooriyama-shi, Fukushima 963–8611, Japan anddDepartment of Pathology, Tochigi Institute of Clinical Pathology, 2308–3 Minamiakatsuka, Nogi, Shimotsuga-gun, Tochigi 329–0112,
Japan(Received September 26, 2008; Accepted October 27, 2008; Published online November 10, 2008)

Nanomaterials are being used increasingly for commercial purposes, yet little is known about the potential
health hazards such materials may pose to consumers and workers. Here we show that nano-sized titanium dioxide(TiO2 ), which is used widely as a photo-catalyst and in consumer products, administered subcutaneously to pregnant mice is transferred to the offspring and affects the genital and cranial nerve systems of the male offspring.
Nanoparticles identified as TiO2 by energy-dispersive X-ray spectroscopy were found in testis and brain of exposed
6-week-old male mice. In the offspring of TiO2 -injected mice, various functional and pathologic disorders, such as
reduced daily sperm production and numerous caspase-3 (a biomarker of apoptosis) positive cells in the olfactory
bulb of the brain, were observed. Our findings suggest the need for great caution to handle the nanomaterials forworkers and consumers.
Key words —— nanoparticle, titanium dioxide (TiO2 ), brain, testis, pregnant mouse, olfactory bulb

INTRODUCTION
Nano-sized particles also known as ultrafine
particles, are very tiny particles less than 100 nm indiameter. They are produced daily by activities suchas driving, cooking, and generating energy in power
plants. Engineered nanomateials are used in sporting goods, tires, stain-resistant clothing, sunscreens,cosmetics, and electonics and will likely be used
increasingly in medicine for purposes of diagnosis
and drug delivery.1–4) Nanotoxicology, the evaluation of the safety of engineered nanostructures andnanodevices, is a novel field of toxicology. Materials that are generally thought to be inert may act
differently when introduced to the body as nanomaterials.4–8)
Nanocrystalline titanium dioxide (TiO2 ), a non∗

To whom correspondence should be addressed: Department ofHygiene Chemistry, Faculty of Pharmaceutical Science, Tokyo
University of Science, 2641 Yamazaki, Noda-shi, Chiba 278–
8510, Japan. Tel.: +81-4-7121-3618; Fax: +81-4-7121-3784;
E-mail:takedak@rs.noda.tus.ac.jp

combustible, odorless powder, is an important material used in commerce. Anatase TiO2 is currently
used in products as diverse as sunscreens and coatings for self-cleaning windows.9) TiO2 can generate reactive oxygen species quite efficiently, particularly when exposed to ultraviolet light. The
photocatalytic activity of the anatase form of TiO2
was reported to be higher than that of the rutile
form.10) Gurr and colleagues11) reported that nanosized anatase TiO2 particles induced oxidative DNA
damage, lipid peroxidation and micronuclei formations and increased hydrogen peroxide and nitric oxide production in BEAS-2B cells, a humanbronchial epithelial cell line, even in the absence ofphotoactivation. However, the potential toxicity of
TiO2 in the next generation has yet to be examined.
In the present study we examined the effects of prenatal exposure to anatase TiO2 on the genital andcranial nerve systems of male offspring mice.

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MATERIALS AND METHODS
Materials —— TiO2 particles (anatase form, particle size 25–70 nm, surface area 20–25 m2 /g, a purity 99.9 %) was purchased from Sigma-Aldrich (StLouis, U.S.A.).
Animals —— Pregnant Slc : ICR mice (purchasedfrom Japan SLC Inc., Shizuoka, Japan) (6 mice/group) received subcutaneous injections of 100 µl of
1 mg/ml TiO2 particles in saline plus 0.05 % Tween
80 at 3, 7, 10, and 14 days postcoitum. Control
mice were treated on the same schedule with 0.05 %Tween 80. Male offspring were weighed and killed
under anesthesia at 4 days or 6 weeks of age. All
experimental animals were handled in accordancewith institutional and national guidelines for thecare and use of laboratory animals.
Organ Weights —— The weights of the testis, epididymis, and seminal vesicle (including prostate,seminal vesicle, and coagulating gland) bilaterally
and brain were measured for each animal, and relative weights (weight of the organ/body weight) were
calculated in 6-week-old offspring.
Daily Sperm Production (DSP) and Morphological Observation of Testis —— Testicular tissue
was thawed and weighed after removal of any extracapsular material from the testis. Testes were
homogenized in buffer containing 0.05 % Triton X100 (Nacalai Tesque, Kyoto, Japan) and 0.2 % Eosin
Y (Merck, Darmstadt, Germany). The number ofsperm nuclei in each suspension was determined byhemocytometer.
Statistical Analysis —— Data were analyzed byMann-Whitney U test, and differences were considered significant at p < 0.05.

Analysis by Field Emission-type Scanning Electron Microscopy (FE-SEM)/Energy-Dispersive
X-ray Spectroscopy (EDS) —— The testis or brain
tissue was embedded in epoxy resin for FESEM/EDS observation. These samples were cut
with thickness of approximately 80 nm with an
Ultra-Microtome (Leica EM UC6rt, Leica Microsystems Japan, Tokyo, Japan). Each ultra-thinsection was placed on a transmission electron microscopy (TEM) grid (Cu 150-B, Okenshoji, Tokyo,
Japan) and analyzed by FE-SEM/EDS (Hitachi
High-technology, Tokyo, Japan).
Methods of Immunohistochemical Staining of
Caspase-3 —— Tissue samples of olfactory fromthe TiO2 treated group and the control group were
fixed with 10 % buffered formalin and, after routine
dehydration, embedded in paraffin. To detect apoptosis in these olfactory under a light microscope,
the immunohistochemical staining for caspase-3 (acommon enzymatic biomarker of apoptosis) was
performed. Paraffin sections 5-µm thick of olfactory samples were stained immunohistochemicallyby the streptoavidin-biotin method (Histofine SABPO kit, Nichirei, Tokyo, Japan). The primary antibody used was anti-human/mouse caspase-3 (active) rabbit IgG (R&D Systems, Inc., Minneapolis,
MN, U.S.A.).

RESULTSTiO2 powder size was confirmed by FE-SEM(Fig. 1). Male offspring were killed under anesthesia at 4 days or 6 weeks of age. In order to determine the genital toxicity of TiO2 particles, bodyFig. 1. Distribution of TiO2 Particle Diameter by FE-SEM(a) FE-SEM Image of TiO2 particles (15.0 kV × 80000, Scale bar, 100 nm). (b) Distribution of TiO2 particle diameters according to FE-SEM
analysis. Columns show the diameter of single particles. Diameter of particles was measured on randomly selected area of FE-SEM image.

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Fig. 2. Detection of TiO2 Nanoparticles in the Testis of Offspring by EDS
Testes were dissected from 6-week-old mice and fixed. Particles were detected in the cells of testis by TEM and field FE-SEM. The particles
were identified as TiO2 by EDS at 7 kV accelerating voltage, 1 × 10−10 A beam current and 100 sec measurement time. Aggregated TiO2 nanoparticles(100–200 nm) were detected in spermatids (a), Sertoli cells (b) and Leydig cells (c). Scale bars, 1 µm. TiO2 particles are indicated by arrows. Particles
in the testis were identified respectively as TiO2 by EDS (d).

and reproduction weights were measured. TiO2 exposed group had significantly lower body weight(88 % relative to control) and significantly higher
weight of epidermis per body weight (117 % relative to control). However, there were no significant
changes in the weight of other reproductive organs.
The presence of TiO2 particles was assessed in
testis and brain from 4-day-old and 6-week-old offspring by TEM and FE-SEM. Particles in the testis
and brain were identified as TiO2 by EDS at 7 kVaccelerating voltage, 1 × 10−10 A beam current, and100 sec measurement time.
As shown in Fig. 2, aggregates of TiO2 nanoparticles (100–200 nm) were detected in Leydig cells,
Sertoli cells, and spermatids in the testis at both
4 days and 6 weeks of age. Sperm samples were collected from the cauda epididymis, and sperm motility and morphology were evaluated under phase
contrast microscopy. Testes of 6-week-old mice
were homogenized, and DSP was examined. Testes
were also fixed and stained with standard procedures for examination by light and electron microscopy.
Among 6-week-old mice, the seminiferous
tubules of hematoxylin and eosin-stained sections

from control mice showed the normal spermatogenic cycle with germ cells and Sertoli cells. Sertoli cells were located regularly in the peripheryof the seminiferous tubules and had large nuclei
with large nucleoli. Testicular morphology in TiO2 exposed mice was abnormal compared to that incontrol mice. In exposed mice, some seminiferous
tubules appeared disorganized and disrupted. There
were fewer mature sperm in the tubule lumen. The
damaged tubules were scattered randomly throughout the testis (Fig. 3). These effects were dependent on the dose of TiO2 and were significantly
higher in the TiO2 exposed mice than in control
mice. DSP per gram of testis, epididymal sperm
motility, and the number of Sertoli cells were significantly lower in mice exposed to TiO2 than incontrol mice. Sperm morphology did not differ significantly (Fig. 4). These data suggest that prenatal
exposure to nano-sized TiO2 has detrimental effects
on mouse spermatogenesis in offspring.
The olfactory bulb and the cerebral cortex(frontal and temporal lobes) of 6-week-old mice
were examined by TEM and FE-SEM/EDS. Nanosized TiO2 particles were detected in cells in brainsof 6-week-old mice exposed prenatally to TiO2

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Fig. 3. Morphology of Seminiferous Tubules and Testicular Functions in 6-week-old Mice Exposed Prenatally to TiO2
Hematoxylin and eosin-stained sections of seminiferous tubules from control mice (a, c) show a normal spermatogenic cycle with germ cells andSertoli cells. Testicular morphology in TiO2 -exposed mice (b, d) was abnormal compared to that in control mice. Some seminiferous tubules appear
disorganized and disrupted. There were fewer mature sperm in the tubule lumen. Damaged tubules were scattered randomly throughout the testis. Scalebars, 100 µm (a, b) and 25 µm (c, d). TEM demonstrating mitochondria (white arrow) of Sertoli cells from control mice (e) and TiO2 -exposed mice (f).
Enlargement of mitochondria and disappearance of cristeae were observed (f). Scale bars, 1 µm (e, f). BM; basement membrane.

(Fig. 5, a–e). We believe that the nanoparticles weretransferred from the mother to the fetus and moved
into the brain because blood-brain barrier was undeveloped.
Numerous cells positive for caspase-3, a common enzymatic marker of apoptosis, were observed
under light microscopy in the olfactory bulb of 6week-old mice exposed prenatally to TiO2 , and thenumber of caspase-3-positive mitral cells was significantly higher in exposed mice than in control
mice (no positive cells, Fig. 6. a, b).
Electron microscopic observations of olfactory
bulb revealed that a subset of cells contained
cresent-shaped spaces (CSS), which are specificfeatures of apoptosis.12) Apoptotic granular perithelial (GP) cells, which are scavenger cells that surround vessels in the brain, contained unidentified
particulate matter. Occlusion of small vessels andperivascular edema were observed in the prenatally
TiO2 -exposed mice.
The abnormalities varied in severity were dependent on the TiO2 concentration, and were notobserved in the control group. These data indicatethat prenatal exposure of mice to TiO2 has a severe
negative effect on fetal brain development and carries a risk of various nervous system disorders.

DISCUSSION
We show here that anatase TiO2 nanoparticles
administered subcutaneously to pregnant mice aretransferred to and affect the genital and cranial
nerve systems of the offspring. These findings suggest that anatase TiO2 can harm the developing fetus
in mice. As we observed in TiO2 -exposed mice, weNo. 1

Fig. 4. Effect of Prenatal Exposure to TiO2 on Seminiferous
Tubles and Testicular Functions in 6-week-old Mice
Testis of 6-week-old mice was homogenized on ice, and DSP wasdetermined (a). Sertoli cells in seminiferous tubules were counted(b). Sperm samples were collected from the cauda epididymis, andmorphology (c) and sperm motility (d) were determined under phase
contrast microscopy. Sertoli cells with damaged mitochondria werecounted by TEM (e). Control: n = 8, TiO2 : n = 8. Presented are themean ± S.E., where ∗ , p < 0.05, ∗∗ , p < 0.01, ∗∗∗ , p < 0.001.

have observed various histologic and functional effects on the male reproductive and central nervous
systems in mice exposed prenatally to diesel exhaust (DE)13–18) and diesel exhaust particles (DEP).
The changes in the reproductive and central nervous systems in DE-exposed mice could be reduced
by eliminating particles including nano-sized particles with a high-quality filter (unpublished data).
Sugamata et al.17) also found that granular perithelial cells, which are scavenger cells, showed signsof apoptosis in the cerebrum and hippocampus ofnewborn mice exposed prenatally to DE. Furthermore, the cytoplasmic granules of these cells contained nano-sized particles. These observations suggest that exposure of pregnant mice to tiny particles
can damage the fetus.
To prevent exposure of the fetus to harmful substances, there is a blood-placenta barrier between
the mother and fetus. There is also a blood-brain
barrier and blood-testis barrier in the important regions of the brain and genitals, respectively, in adult99

mice. Our present electron microscopy data indicate that nanoparticles can transfer from pregnant
mice into brain and testis of their offspring. These
blood barriers are undeveloped or under developedin the fetus, therefore, harmful nanoparticles could
easily pass into the brain during the early stages offetal development.
Nano-sized particles can enter the human body
via the lungs and intestines. Whether such particles
can penetrate the skin is less clear,6, 7) Kreilgaard19)
suggested that very small TiO2 particles (e.g. 5–
20 nm) can penetrate the skin and interact with the
immune system. Tinkle et al.20) showed that 0.5and 1.0-µm particles, in conjunction with motion,
penetrate the stratum corneum of human skin andreach the epidermis and, occasionally, the dermis.
There are reports that inhaled or injected
nanoparticles enter the systemic circulation21–23)
and migrate to various organs and tissues.24) If particles enter the body, their distribution is a function of their size and surface characteristics. Theremay be a critical size beyond which movement of
the nanoparticles within the body is restricted. The
brain is especially vulnerable to oxygen stress damage, and recent studies have supported our present
and previous findings that nanosized particles canbe uptaken in brain25) and enter the central nervous
system.26) Oberdörster et al.27) reported that inhaled
nanoparticles could be translocated into brain via
the olfactory nerves. Sugamata et al.18) reported
previously that specific features of apoptosis were
present in Purkinje cells of cerebellum in mice exposed prenatally to DE. In the present study, we observed few apoptotic features in Purkinje cells of
TiO2 -exposed mice. DEP and TiO2 particles may
differ in their abilities to induce apoptosis in cerebellum.
Regardless of the particle size, TiO2 has only
minimal effects in adult rodents.28) However, numerous in vitro studies revealed that TiO2 nanoparticles cause oxidative stress-mediated toxicity indiverse cell types including skin fibroblasts,29)
alveolar macrophages.30) Long et al.31) showedthat mouse microglia engulfed the TiO2 particles
and, for 2 hr, released bursts of reactive oxygen
molecules that interfered with mitochondrial energy
production. This did not damage the microglia,
however, prolonged exposure to such compounds
can damage neurons. Greater surface area per massrenders nano-size particles more active biologicallythan larger particles of the same chemical makeup.
Numerous studies regarding the effects of ul-

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Fig. 5. Detection of TiO2 Nanoparticles in the Olfactory Bulb and Cerebral Cortex of Brain of Offspring of TiO2 -exposed Mice by
EDS
Olfactory bulb and cerebral cortex were dissected from 6-week-old mice and fixed. Particles were detected by TEM and FE-SEM. Photographsdemonstrating aggregated TiO2 nanoparticles (100–200 nm) in endotherial cells of olfactory bulb (a), and nerve cell fibers in cerebral cortex (c). Scalebars, 1 µm. TiO2 particles are indicated by arrows. Particles in the brain were identified respectively as TiO2 by EDS at 15 kV (b) and 7 kV (d)accelerating voltage, 1 × 10−10 A beam current and 100 sec measurement time. Electron micrograph demonstrating magnified aggregated TiO2 particles
in nerve cells in cerebral cortex (e).

Fig. 6. Immunohistochemical Staining of Caspase-3 in Olfactory Bulb of 6-week-old Mice(a) Control mice, (b) mice exposed prenatally to TiO2 . Numerous caspase-3 positive mitral cells are visible and the number of positive cells in
TiO2 -exposed mice is significantly higher compared with that in control mice.

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trafine particle pollutants on respiratory and circulatory systems have been reported. However, little is known about the effect on the genital andcentral nervous systems. Our present and former
findings suggest that widespread use of TiO2 andother nanoparticles including ultrafine particulates
in air might affect unborn children, especially development of their reproductive and nervous systems. Therefore, research into the risk of exposure
to nanoparticles, into removal of nanoparticles fromthe environment, and into methods to protect against
toxicity of such particles is important.
Acknowledgements We thank Dr. I. Sugawara of
Research Institute of Tuberculosis for help withpreparation of testis samples for histology. We
thank the graduate and undergraduate students in
the Takeda and Nihei laboratories. This work was
supported in part by a Grant-in-Aid for Science
Research from the Ministry of Education, Culture,
Sports, Science and Technology of Japan, a Grantin Aid for the Private University Science ResearchUpgrade Promotion Business Academic Frontier
Project and a Grant-in Aid for Health and Labour
Sciences Research Grants, Research on Risk ofChemical Substances, from the Ministry of Health,Labour and Welfare.

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