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 . Tin-Tin-Win-Shwe et al.  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.  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  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 , 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 . A subsequent report  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 , 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 , and glial cell line-derived neurotrophic factor , and hormones including growth hormone  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.  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 . 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 . 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" . 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  and unpublished data suggest that other organ systems are also affected.