Maternal exposure to nanoparticulate titanium dioxide during the prenatal period alters gene expression related to brain development in the mouse

  • Midori Shimizu1,

    Affiliated with

    • Hitoshi Tainaka2,

      Affiliated with

      • Taro Oba1,

        Affiliated with

        • Keisuke Mizuo2,

          Affiliated with

          • Masakazu Umezawa1 and

            Affiliated with

            • Ken Takeda1, 2Email author

              Affiliated with

              Particle and Fibre Toxicology20096:20

              DOI: 10.1186/1743-8977-6-20

              Received: 1 April 2009

              Accepted: 29 July 2009

              Published: 29 July 2009



              Nanotechnology is developing rapidly throughout the world and the production of novel man-made nanoparticles is increasing, it is therefore of concern that nanomaterials have the potential to affect human health. The purpose of this study was to investigate the effects of maternal exposure to nano-sized anatase titanium dioxide (TiO2) on gene expression in the brain during the developmental period using cDNA microarray analysis combined with Gene Ontology (GO) and Medical Subject Headings (MeSH) terms information.


              Analysis of gene expression using GO terms indicated that expression levels of genes associated with apoptosis were altered in the brain of newborn pups, and those associated with brain development were altered in early age. The genes associated with response to oxidative stress were changed in the brains of 2 and 3 weeks old mice. Changes of the expression of genes associated with neurotransmitters and psychiatric diseases were found using MeSH terms.


              Maternal exposure of mice to TiO2 nanoparticles may affect the expression of genes related to the development and function of the central nervous system.


              Nanotechnology and the production of novel man-made nanoparticles are increasing worldwide. Titanium dioxide (TiO2) has a high level of photocatalytic activity, and can be used for air and water purification and self-cleaning surfaces [1]. The activity level of nanoparticles is higher than that of bulk-sized particles [2, 3]. TiO2 has the potential to produce reactive oxygen species (ROS) in its photocatalysis [1] and its possibly detrimental health effects are of concern. It has been reported that a mixture of anatase and rutile TiO2 nanoparticles induced cytotoxicity against human lung epithelial cells (BEAS-2B), even in the absence of photoactivation [4]. Sayes et al. [5] showed that anatase TiO2 nanoparticles, which can generate more ROS than rutile TiO2 particles, exhibited a higher level of cytotoxicity against human dermal fibroblasts and human lung epithelial cells (A549) than rutile TiO2 nanoparticles.

              The small size of nanoparticles can bestow unique translocational properties [6, 7]. It has been reported that nanosized elemental carbon particles (36 nm) inhaled by adult rats were translocated into extrapulmonary organs, such as liver [8]. A subsequent study showed that intranasally instilled carbon black nanoparticles can be translocated to the central nervous system, including cerebrum, cerebellum, and olfactory bulb via the olfactory nerve [9]. In a recent study, Takeda et al. [10] found that TiO2 nanoparticles administrated subcutaneously to pregnant mice were transferred from the mother to the fetal brain, and induced apoptosis in the mitral cells of the olfactory bulb of mice exposed maternally to the nanoparticles. Fetal brains are easily affected by blood-borne substances, including nano-sized materials, to a much greater extent than adult brains because the development of the blood-brain barrier in the fetal brains is incomplete [11]. Taking these observations into consideration, functional alterations of the central nervous system induced by maternal exposure to nanoparticles need to be investigated. To analyze the effect of maternal exposure to TiO2 nanoparticles on the early stages of development of the brain, we used microarray technology and gene expression profiles by functional annotation of genes using Gene Ontology (GO) terms and Medical Subject Headings (MeSH) terms.


              Titanium dioxide nanoparticles

              TiO2 nanopowder (particle size 25-70 nm; surface area 20-25 m2/g; crystal form anatase) was purchased from Sigma-Aldrich Japan Inc. (Tokyo, Japan) and used as TiO2 nanoparticles. The nanopowder was suspended in saline (Otsuka Pharmaceutical Factory Inc., Tokushima, Japan) with 0.05% (v/v) Tween 80 and sonicated for more than 30 minutes immediately before administration.

              Animals and treatments

              Pregnant ICR mice, purchased from Japan SLC Inc. (Shizuoka, Japan), were housed in a room under controlled temperature (23 ± 1°C), humidity (55 ± 5%) and light (12 h light/12 h dark cycle with light on at 8:00 a.m.) with ad libitum access to food and water. Pregnant mice were transported carefully to minimize stress factors by Sankyo Labo Service Co., Inc (Tokyo, Japan). All animals were handled in accordance with institutional and national guidelines for the care and use of laboratory animals.

              A 100 μL volume of TiO2 suspended at 1 μg/μL was injected subcutaneously into pregnant mice (n = 15) on gestational days 6, 9, 12, and 15 for the exposure group, while 100 μL of vehicle alone was injected into pregnant mice (n = 14) as a control group. Brain tissue was obtained from male fetuses on embryonic day (ED) 16 (n = 8/group) and from male pups on postnatal days 2 (n = 10/group), 7 (n = 10/group), 14 (n = 9/group), and 21 (n = 9/group).

              Total RNA extraction

              Whole brains were immediately frozen in liquid nitrogen and kept at -80°C. Frozen tissue was homogenized and extracted with Isogen (Nippon Gene Co., Ltd., Tokyo, Japan) while well stirred by a Vortex-Genie 2 (Scientific Industries, Tokyo, Japan). Total RNA was isolated according to the manufacture's protocol and suspended in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).

              Complementary DNA microarray analysis

              RNAs for microarray analysis were pooled for each group, purified using the RNeasy Micro Kit (Qiagen, Hilden, Germany) and reverse-transcribed to yield complementary DNA (cDNA) labeled with the fluorescent dye Cy3 or Cy5 using the SuperScript Indirect cDNA Labeling Core Kit (Invitrogen, CA, USA) and the SuperScript Indirect cDNA Labeling System Purification Kit (Invitrogen). Cy3- and Cy5-labeled samples were purified using the CyScribe GFX Purification Kit (GE Healthcare Bio-Sciences, Little Chalfont, UK). The generated targets were mixed and subjected to hybridization to an NIA mouse 15 K Microarray v2.0 (AGC Techno Glass Co. Ltd., Chiba, Japan) consisting of 16,192 gene probes. Microarrays were scanned with two different photomultiplier sensitivities by a ScanArray (Packard BioChip Technologies, MA, USA). The scanner output images were normalized and signal quantification was performed using ScanArray Express (Perkin Elmer, MA, USA) and TIBCO Spotfire (TIBCO Software Inc., CA, USA). Normalization was used so that the overall intensity ratio of Cy3 and Cy5 was equal to 1. Statistical analysis was done with analysis of variance (ANOVA) and the level of statistical significance was set at P < 0.05.

              Functional analysis of microarray data with gene annotation

              A total of 37 GO terms and 66 MeSH terms associated with anatomy, brain development and associated peptides, neurotransmitters, hormones, behavior and psychological phenomena, brain related disorders, oxidative stress, inflammation, and cell death were selected (Table 1, 2); and 2838 and 3625 genes were annotated by GO and MeSH terms, respectively, using the gene reference database PubGene (https://​server.​pubgene.​com/​online/​PubGene/​, Pub Gene AS, Oslo, NOR). These annotations were updated in April, 2008. The genes for which upregulation and downregulation were detected were categorized with GO and MeSH terms. The enrichment factor for each category was defined as (nf/n)/(Nf/N), where nf is the number of differentially expressed genes within the category, n is the total number of genes within that same category, Nf is the number of differentially expressed genes on the entire microarray, and N is the total number of genes on the microarray. Statistical analysis was performed using Fisher's exact test with hypergeometric distribution and the level of statistical significance was set at P < 0.05.
              Table 1

              List of GO terms selected for gene annotation



              GO term

              biological process

              developmental process

              brain development


                 forebrain development


                 midbrain development


                 hindbrain development


              generation of neurons


              glial cell differentiation


              biological regulation

              cell death




              neuron apoptosis


              activated T cell apoptosis


              B cell apoptosis


              negative regulation of neuron apoptosis


              apoptotic mitochondrial changes


                 induction of programmed cell death


              induction of apoptosis




              glucocorticoid biosynthesis


              glucocorticoid metabolism


              neurotransmitter metabolism


              neurotransmitter transport


              multicellular organismal process



              learning and, or memory


              regulation of biological process

              regulation of glial cell differentiation


              regulation of nerve growth factor receptor activity


              regulation of glucocorticoid biosynthesis process


              cellular process

              mitochondrial fission


              mitochondrial fusion


              response to stimulus

              response to oxidative stress


                 response to reactive oxygen species


              response to superoxide


              superoxide metabolism


              glutathione biosynthesis


              glutathione metabolism

              molecular function


              motor activity


              superoxide dismutase activity


              glucocorticoids receptor activity


              brain derived neurotrophic factor binding

              Table 2

              List of MeSH terms selected for gene annotation


              MeSH term



              Blood Brain Barrier




              Olfactory Receptor Neurons








              Alzheimer Disease



              Anxiety Disorders

              Learning Disorders


              Attention Deficit Disorder

              Memory Disorders


              with Hyperactivity

              Mitochondrial Disease


              Autistic Disorder

              Neurogenic Inflammation


              Cognition Disorders

              Parkinson Disease




              Psychiatry and Psychology

              Affective Symptoms




              Memory, Short-Term






              Stress, Psychological




              Chemicals and Drugs

              Apoptosis Inducing Factor

              Anti-Anxiety Agents


              Apoptosis Regulatory Proteins




              Glutathione Peroxidase


              Brain Derived Neurotrophic

              Glutathione Synthase



              Inflammation Mediators


              Glial Cell Line-Derived

              Neuronal Apoptosis-


              Neurotrophic Factor

              Inhibitory Protein


              Nerve Growth Factor

              Nitric Oxide



              Reactive Oxygen Species





              Growth Hormone

              Superoxide Dismutase


              Thyroid Hormones












              gamma-Aminobutyric Acid



              Glutamic Acid



              Neurotransmitter Uptake



              Biological Science


              Motor Activity


              Cell Death

              Neural Plasticity


              Gene, Mitochondrial

              Oxidative Stress


              Lipid Peroxides



              Analysis of cDNA microarrays

              In the maternal TiO2 exposure group, the expression levels of 462 genes were changed significantly in the brain of the fetus at ED 16 (upregulation 229 genes; downregulation 233 genes), and those of 864 (upregulation 234; downregulation 630), 417 (upregulation 351; downregulation 66), 738 (upregulation 450; downregulation 288), and 1887 (upregulation 613; downregulation 1274) were changed significantly in the brain of offspring 2, 7, 14, and 21 days old, respectively (Table 3). The number of genes differentially expressed between groups was increased remarkably in the brain of 21 days old pups.
              Table 3

              The number of genes differentially expressed in maternal TiO2 exposure group





              Embryonic day 16




              2 days old




              7 days old




              14 days old




              21 days old




              Functional categorization of microarray data

              Of the genes expressed differentially in the maternal TiO2 exposure group, 3, 2, 8, and 4 GO categories were enriched significantly in the brain at 2, 7, 14, and 21 days after birth, respectively (Table 4), while 6, 2, 36, and 28 MeSH categories were enriched significantly at 2, 7, 14, and 21 days after birth (Additional file 1). Eight MeSH categories were also enriched significantly in the fetal brain at ED 16 (Additional file 1). The largest group of GO categories enriched was those related to cell death 2 - 21 days after birth; 121 and 64 genes linked to apoptosis at 2 and 7 days after birth, respectively, and 92 and 173 genes linked to "cell death" were identified at 14 and 21 days after birth. "Brain development" was also a large category at 2 (34 genes) and 14 (43 genes) days after birth. GO categories related to oxidative stress, such as "superoxide dismutase activity", were also enriched significantly at 14 and 21 days after birth. The largest MeSH categories enriched were "Mitochondria" at ED 16 (31 genes) and 2 days (56 genes) after birth and "Apoptosis" at 14 (118 genes) and 21 (230 genes) days after birth. The "Mitochondria" category was persistently enriched at 14 (60 genes) and 21 (109 genes) days after birth. MeSH categories related to oxidative stress, such as "Glutathione", "Lipid Peroxidation", and "Reactive Oxygen Species", were also enriched significantly at ED 16 and 14 and 21 days after birth. MeSH categories related to inflammation and neurotransmitters including "Epinephrine", "Norepinephrine", "Serotonin", and "Glutamic Acid" were also highly enriched at 14 and 21 days after birth.
              Table 4

              Significantly enriched GO categories in maternal exposure group vs. control group

              GO term

              Enrichment factor

              P value

              Embryonic day 16




              2 days old





                 brain development



                 motor activity



              7 days old





                 glial cell differentiation



              14 days old


                 activated T cell apoptosis



                 brain development



                 cell death



                 induction of apoptosis



                 motor activity



                 response to oxidative stress



                 response to reactive oxygen species



                 superoxide dismutase activity



              21 days old





                 cell death



                 glutathione biosynthesis



                 superoxide dismutase activity




              Nanoparticles have a high level of reactivity with biological tissue, since they have a large specific surface area [6, 7]. It has been reported that fullerenes, which are manufactured carbon nanoparticles, induce oxidative stress in the brain of juvenile largemouth bass [12]. Tin-Tin-Win-Shwe et al. [13] showed that intranasal instillation of ultrafine carbon black (14 nm) to mice induced a higher level of expression of cytokines and chemokines in the olfactory bulb compared to those induced by the same mass of carbon black (95 nm). The particles used in the exposed pregnant mice group can enter the circulatory system and can transfer to and damage the fetus. Sugamata et al. [14] reported that the cytoplasmic granules of granular perithelial cells contain particles of diesel exhaust (DE) and degenerate in both the cerebral cortex and the hippocampus of mice exposed prenatally to DE. A later study [15] showed that maternal DE exposure alters the levels of monoamines and their metabolites in brains and spontaneous motor activity in male mice. Since TiO2 was detected in the brain of mice maternally exposed to TiO2 nanoparticles [10], which is the material used in this study, microarray was applied to the analysis of the effects of maternal exposure to TiO2 nanoparticles on the brain of neonatal mice.

              In the present study, we used only male fetuses and pups for analysis because the prevalence of some psychiatric disorders in childhood, such as autism and attention deficit hyperactivity disorder, is higher in men than in women. The results of the microarray analysis showed changes in expression of hundreds of genes in the brain at ED 16, and at 2, 7, 14, and 21 days after birth. To interpret the large amount of data generated, functional categorization using GO terms and MeSH terms were performed, which identified potentially important categories on the basis of both a high enrichment factor (>1.00) and statistical significance (P < 0.05). MeSH is a controlled vocabulary thesaurus produced by the National Library of Medicine and used for indexing, cataloging, and searching for biomedical and health-related information and documents. Although most researchers use GO for providing annotation to genes, MeSH terms are proposed to be a useful complementary tool for interpretation of microarray data [16]. A subsequent report [17] showed that the use of MeSH has the advantage of producing anatomical and disease information with respect to the genes of interest. In the present study, genes were annotated with the terms related to anatomy, brain development, brain-related disorders, those associated with nanotoxicology (oxidative stress [6, 7, 12] and inflammation [6, 7, 13]), and those associated with the effects of maternal exposure to DE or TiO2 nanoparticles (hormones [18], behavior and neurotransmitters [15, 18], and cell death [10, 14, 19]) for analysis.

              As a result, GO terms associated with development of brain were extracted at 2 and 14 days after birth, those associated with cell death, including apoptosis, were extracted 2 to 21 days after birth, and those associated with response to oxidative stress were extracted at 14 and 21 days. Brain development is regulated by neurotrophins such as nerve growth factor, brain-derived neurotrophic factor [20], and glial cell line-derived neurotrophic factor [21], and hormones including growth hormone [22] and thyroid hormone [23, 24]. Analysis using MeSH terms showed that alteration of these factors that can lead to abnormal development of the central nervous system was induced by maternal exposure to TiO2 nanoparticle. It has been reported that neuronal cell death, including apoptosis, is essential for elimination of neurons and axons to make correct synaptogenesis in the early stage of brain development [25, 26]. The result of functional analysis suggested that disruption of these processes can be caused by maternal exposure to TiO2 nanoparticles.

              It has been reported that the changes of environment surrounding pregnant mice cause abnormal level of neurotransmitters in the brain of the offspring. Meyer et al. [27] reported that maternal immune challenge by the viral mimic polyriboinosinic-polyribocytidilic acid causes abnormal fetal dopaminergic development, which is similar to a schizophrenic symptom. Maternal stress also induces altered expression of genes related to the dopaminergic system in the midbrain and causes hyperactivity in adult offspring [28]. The results that MeSH terms associated with neurotransmitters and motor activity were extracted suggest that maternal exposure to TiO2 nanoparticles causes abnormal levels of neurotransmitters that can lead to altered motor activity.

              As for MeSH terms, those associated with diseases were extracted in the functional analysis. Some diseases such as autistic disorder, epilepsy, and learning disorders, occur in childhood, and although Alzheimer's disease, schizophrenia, and Parkinson's disease arise mainly in adulthood or old age, related MeSH terms were extracted in the results from infant mice of mothers exposed to TiO2. In the early 1990s, Dr David Barker J.P. stated that fetal undernutrition increases the incidence of cardiovascular disease in adult life [29]. Subsequent studies showed the environment that the fetus senses indirectly through the mother can be linked to other diseases in adulthood, and proposed a hypothesis of "early developmental origins of adult disease" [30]. The results of the present study suggest that maternal exposure to nanoparticles can alter gene expression in the neonatal period and might cause the onset of psychiatric disorders even in adulthood. However, the present study did not show how the maternal response to the nanoparticles altered the mother's behavior toward the pups and how this in turn altered gene expression. Further investigations are needed to clarify the critical factor for the gene expression change. Moreover, the changes caused by maternal exposure to TiO2 nanoparticles should not be limited to the brain. Our published [10] and unpublished data suggest that other organ systems are also affected.


              This study showed that maternal exposure to anatase TiO2 nanoparticle caused the changes in the expression of genes associated with brain development, cell death, response to oxidative stress, and mitochondria in the brain during the perinatal period, and those associated with inflammation and neurotransmitters in the later stage (Figure 1). Further investigation is needed to clarify the alterations of neurotransmitter levels and motor function. This study showed also that analysis using microarray data with GO and MeSH terms can provide meaningful information, and will contribute to further interpretation of microarray results in toxicological research.
              Figure 1

              Summary of the extracted terms with genes differentially expressed in the maternal TiO 2 exposure group.



              complementally DNA


              diesel exhaust


              embryonic day


              Gene Ontology


              Medical Subject Headings


              reactive oxygen species


              titanium dioxide.



              The authors thank Dr Tomomi Hishinuma of Research Center for Health Sciences of Nanoparticles, Tokyo University of Science for assistance in the microarray experiments, and Drs Masao Sugamata and Tomomi Ihara of Tochigi Institute of Clinical Pathology for valuable discussion. 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 Gland-in Aid for the Private University Science Research Upgrade Promotion Business "Academic Frontier Project and a Grant-in Aid for Health and Labour Sciences Research Grants, Research on Risk of Chemical Substances, from the Ministry of Health, Labour and Welfare".

              Authors’ Affiliations

              Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science
              Research Center for Health Sciences of Nanoparticles, Research Institute for Science and Technology, Tokyo University of Science


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              © Shimizu et al; licensee BioMed Central Ltd. 2009

              This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.