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.

Results: 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.

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

The small size of nanoparticles can bestow unique trans­locational properties . It has been reported that nano-sized elemental carbon particles (36 nm) inhaled by adult rats were translocated into extrapulmonary organs, such as liver. 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 . In a recent study, Takeda et al.  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 . 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.

Methods

Titanium dioxide nanoparticles

TiO₂ nanopowder (particle size 2570 nm; surface area 2025 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

Figure 1. Summary of the extracted terms with genes differentially expressed in the maternal TiO₂ exposure group.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 ; 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.

(Midori Shimizu , Hitoshi Tainaka , Taro Oba , Keisuke Mizuo , Masakazu Umezawa and Ken Takeda, Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba 278-8510, Japan and Research Center for Health Sciences of Nanoparticles, Research Institute for Science and Technology, Tokyo University of Science, Yamazaki 2641, Noda-shi, Chiba 278-8510, Japan)