Masakazu Umezawa

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

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

In utero exposure to a low concentration of diesel exhaust affects spontaneous locomotor activity and monoaminergic system in male mice

BackgroundEpidemiological studies have suggested that suspended particulate matter (SPM) causes detrimental health effects such as respiratory and cardiovascular diseases, and that diesel exhaust particles from automobiles is a major contributor to SPM. It has been reported that neonatal and adult exposure to diesel exhaust damages the central nervous system (CNS) and induces behavioral alteration. Recently, we have focused on the effects of prenatal exposure to diesel exhaust on the CNS. In this study, we examined the effects of prenatal exposure to low concentration of diesel exhaust on behaviour and the monoaminergic neuron system. Spontaneous locomotor activity (SLA) and monoamine levels in the CNS were assessed.MethodsMice were exposed prenatally to a low concentration of diesel exhaust (171 μg DEP/m3) for 8 hours/day on gestational days 2-16. SLA was assessed for 3 days in 4-week-old mice by analysis of the release of temperature-associated infrared rays. At 5 weeks of age, the mice were sacrificed and the brains were used for analysis by high-performance liquid chromatography (HPLC).Results and DiscussionMice exposed to a low concentration of diesel exhaust showed decreased SLA in the first 60 minutes of exposure. Over the entire test period, the mice exposed prenatally to diesel exhaust showed decreased daily SLA compared to that in control mice, and the SLA in each 3 hour period was decreased when the lights were turned on. Neurotransmitter levels, including dopamine and noradrenaline, were increased in the prefrontal cortex (PFC) in the exposure group compared to the control group. The metabolites of dopamine and noradrenaline also increased in the PFC. Neurotransmitter turnover, an index of neuronal activity, of dopamine and noradrenaline was decreased in various regions of the CNS, including the striatum, in the exposure group. The serum corticosterone level was not different between groups. The data suggest that decreased SLA in mice exposed prenatally to diesel exhaust is due to facilitated release of dopamine in the PFC.ConclusionsThese results indicate that exposure of mice in utero to a low concentration of diesel exhaust decreases SLA and alters the neurochemical monoamine metabolism of several regions of the brain.

Cytokine and chemokine expression in a rat endometriosis is similar to that in human endometriosis.

The pathogenesis of endometriosis, a gynecologic disorder associated with infertility, appears to involve immune responses. However, the details involved have not been clarified. In this study, we analyzed expression levels of interleukin (IL)-6, IL-10, monocyte chemoattractant protein-1, eosinophil chemotactic protein, macrophage inflammatory protein-1alpha, and regulated on activation normal T cell expressed and secreted (RANTES) and CC chemokine receptor 1 in endometriotic lesions in a rat model in which endometrium is autotransplanted onto peritoneal tissue and found that they were remarkably increased, while those of IL-2, IL-4, and interferon-gamma were not. These results were obtained in a rat model induced by autologous, not allogeneic, transplantation of endometrial epithelium to the peritoneum. Expression of these factors is consistent with that of endometriosis in humans. Therefore, this model may be useful in the investigation of the pathogenesis and treatment of endometriosis.

Carbon black nanoparticle exposure during middle and late fetal development induces immune activation in male offspring mice

Increasing exposure to nanoparticles (NPs) has raised concerns regarding their health and safety profiles in humans and animals, especially in developing organisms, which may display increased sensitivity to NP toxicity. The present study examined the effects of gestational exposure to carbon black NP (CB-NP) on the development of the offspring immune system. Pregnant mice were exposed to CB-NP (95μg/kg body weight) by intranasal instillation on gestational days 9 and 15. The thymus and spleen were collected from their offspring mice on postnatal day (PND) 1, 3 and 5. Thymocyte and splenocyte phenotypes were examined by determining the expression of cell-surface molecules using flow cytometry. Gene expression in the thymus and spleen was examined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Prenatal exposure to CB-NP increased total thymocytes and their immunophenotypes (CD4(-)CD8(-) and CD4(+)CD8(+) cells). It also induced an increase in total lymphocytes, and CD4(-)CD8(-), particularly CD3(-)B220(-)cells, at PND 5 in the spleen of newborn male offspring, reflecting the stimulation of immature splenocytes. Furthermore, mRNA expression of genes related to the induction of peripheral tolerance (i.e. thymic Traf6) was upregulated. These data suggest that respiratory exposure to CB-NP during middle and late gestation may have allergic or inflammatory effects in male offspring, and may provide initial information on the potential developmental immunotoxicity of nanoparticles.

Effects of Maternal Exposure to Ultrafine Carbon Black on Brain Perivascular Macrophages and Surrounding Astrocytes in Offspring Mice

Perivascular macrophages (PVMs) constitute a subpopulation of resident macrophages in the central nervous system (CNS). They are located at the blood-brain barrier and can contribute to maintenance of brain functions in both health and disease conditions. PVMs have been shown to respond to particle substances administered during the prenatal period, which may alter their phenotype over a long period. We aimed to investigate the effects of maternal exposure to ultrafine carbon black (UfCB) on PVMs and astrocytes close to the blood vessels in offspring mice. Pregnant mice were exposed to UfCB suspension by intranasal instillation on gestational days 5 and 9. Brains were collected from their offspring at 6 and 12 weeks after birth. PVM and astrocyte phenotypes were examined by Periodic Acid Schiff (PAS) staining, transmission electron microscopy and PAS-glial fibrillary acidic protein (GFAP) double staining. PVM granules were found to be enlarged and the number of PAS-positive PVMs was decreased in UfCB-exposed offspring. These results suggested that in offspring, “normal” PVMs decreased in a wide area of the CNS through maternal UfCB exposure. The increase in astrocytic GFAP expression level was closely related to the enlargement of granules in the attached PVMs in offspring. Honeycomb-like structures in some PVM granules and swelling of astrocytic end-foot were observed under electron microscopy in the UfCB group. The phenotypic changes in PVMs and astrocytes indicate that maternal UfCB exposure may result in changes to brain blood vessels and be associated with increased risk of dysfunction and disorder in the offspring brain.

Microarray analysis provides insight into the early steps of pathophysiology of mouse endometriosis model induced by autotransplantation of endometrium

AIMS To characterize the biochemical alterations that occur in the peritoneal tissue of the mouse endometriosis model during early development of the lesion using microarray analysis. MAIN METHODS The endometriosis model was induced by autotransplantation of endometrium in 8-week-old female ICR mice. Peritoneum only (excluding the transplant) was obtained 24, 48, and 96 h after the autotransplantation and subjected to microarray analysis. To interpret the large amounts of data generated and to enable a functional analysis, genes were classified using Gene Ontology (GO) and Medical Subject Heading (MeSH) terms, and the results were compared with previous reports on endometriosis. KEY FINDINGS Of the upregulated genes, those involved in the inflammatory response, cell adhesion, extracellular matrix, wound healing, hormones, and leukocytes were significantly enriched 24 and 48 h after autotransplantation. Those of cytokines, antibody-producing cells, dendritic cells, inflammation, and infertility were enriched after 96 h. Analysis using GO and MeSH provided different information. Particularly, MeSH showed a link between an anatomical and diseased phenotype with common genes found to be upregulated. SIGNIFICANCE The factors occurring during early development of endometriosis induced by endometrium autotransplantation are increase in adhesion molecules and inflammatory responses rather than angiogenesis. Data presented herein may reveal a novel therapeutic gene targets and will contribute to knowledge for the treatment of this currently incurable disease.

Gene Expression Changes in the Olfactory Bulb of Mice Induced by Exposure to Diesel Exhaust Are Dependent on Animal Rearing Environment

There is an emerging concern that particulate air pollution increases the risk of cranial nerve disease onset. Small nanoparticles, mainly derived from diesel exhaust particles reach the olfactory bulb by their nasal depositions. It has been reported that diesel exhaust inhalation causes inflammation of the olfactory bulb and other brain regions. However, these toxicological studies have not evaluated animal rearing environment. We hypothesized that rearing environment can change mice phenotypes and thus might alter toxicological study results. In this study, we exposed mice to diesel exhaust inhalation at 90 µg/m3, 8 hours/day, for 28 consecutive days after rearing in a standard cage or environmental enrichment conditions. Microarray analysis found that expression levels of 112 genes were changed by diesel exhaust inhalation. Functional analysis using Gene Ontology revealed that the dysregulated genes were involved in inflammation and immune response. This result was supported by pathway analysis. Quantitative RT-PCR analysis confirmed 10 genes. Interestingly, background gene expression of the olfactory bulb of mice reared in a standard cage environment was changed by diesel exhaust inhalation, whereas there was no significant effect of diesel exhaust exposure on gene expression levels of mice reared with environmental enrichment. The results indicate for the first time that the effect of diesel exhaust exposure on gene expression of the olfactory bulb was influenced by rearing environment. Rearing environment, such as environmental enrichment, may be an important contributive factor to causation in evaluating still undefined toxic environmental substances such as diesel exhaust.

Ratiometric near-infrared fluorescence nanothermometry in the OTN-NIR (NIR II/III) biological window based on rare-earth doped β-NaYF4 nanoparticles

A novel nanothermometer based on over-1000 nm (OTN) near-infrared (NIR) emission of rare-earth doped ceramic nanophosphors (RED-CNPs) was developed for temperature measurement in deep tissue. Hexagonal-phase β-NaYF4 nanoparticles co-doped with Yb3+, Ho3+, and Er3+ (NaYF4:Yb3+,Ho3+,Er3+ NPs) were synthesized and used as a nanothermometer. The NaYF4:Yb3+,Ho3+,Er3+ NPs displayed two OTN-NIR emission peaks in the second (NIR-II) (at 1150 nm of Ho3+) and third (NIR-III) (at 1550 nm of Er3+) biological window regions under NIR (980 nm) excitation in the first (NIR-I) biological window region. Oleic acid (OA) capped NaYF4:Yb3+,Ho3+,Er3+ NPs were dispersed in non-polar media, i.e., cyclohexane, and showed a temperature-dependent intensity ratio of the emission peaks of Ho3+ and Er3+ (IHo/IEr). The temperature-dependent IHo/IEr of the OA-NaYF4:Yb3+,Ho3+,Er3+ NPs was also evident through imitation tissue. The surfaces of the NaYF4:Yb3+,Ho3+,Er3+ NPs were modified with a poly(ethylene glycol) (PEG)-based block copolymer. The PEGylated NaYF4:Yb3+,Ho3+,Er3+ NPs were dispersed in water and emitted strong NIR-II and III emissions under NIR-I excitation. The PEGylated NaYF4:Yb3+,Ho3+,Er3+ NPs were injected into mice via the tail vein, and the OTN-NIR emissions of the PEGylated NaYF4:Yb3+,Ho3+,Er3+ NPs from the mouse blood vessels were clearly observed using an OTN-NIR fluorescence in vivo imaging system. In a polar media, water, the IHo/IEr of PEGylated NaYF4:Yb3+,Ho3+,Er3+ NPs was inversely related to the temperature. In both non-polar and polar media, the IHo/IEr values of the NaYF4:Yb3+,Ho3+,Er3+ NPs were almost linearly dependent on the temperature. The obtained NaYF4:Yb3+,Ho3+,Er3+ NPs are promising as a novel fluorescent nanothermometer for deep tissue.

In utero exposure of mice to diesel exhaust particles affects spatial learning and memory with reduced N-methyl-D-aspartate receptor expression in the hippocampus of male offspring.

Diesel exhaust consists of diesel exhaust particles (DEPs) and gaseous compounds. Previous studies reported that in utero exposure to diesel exhaust affects the central nervous system. However, there was no clear evidence that these effects were caused by diesel exhaust particles themselves, gaseous compounds, or both. Here, we explored the effects of in utero exposure to DEPs on learning and memory in male ICR mice. DEP solutions were administered subcutaneously to pregnant ICR mice at a dose of 0 or 200 μg/kg body weight on gestation days 6, 9, 12, 15, and 18. We examined learning and memory in 9-to-10-week-old male offspring using the Morris water maze test and passive avoidance test. Immediately after the behavioral tests, hippocampi were isolated. Hippocampal N-methyl-D-aspartate receptor (NR) expression was also measured by quantitative RT-PCR analysis. Mice exposed to DEPs in utero showed deficits in the Morris water maze test, but their performance was not significantly different from that of control mice in the passive avoidance test. In addition, DEP-exposed mice exhibited decreased hippocampal NR2A expression. The present results indicate that maternal DEP exposure disrupts learning and memory in male offspring, which is associated with reduced hippocampal NR2A expression.

Expression Profile of Extracellular Matrix and Adhesion Molecules in the Development of Endometriosis in a Mouse Model

Ectopic endometrial tissue induces various reactions in surrounding tissues, such as the surface of the ovary and peritoneal cavity, leading to endometriosis. The aim of this study is to investigate the expression profile of extracellular matrix (ECM) and adhesion molecules in the early steps of development of experimental mouse endometriosis, specifically in peritoneum adjacent to endometrium transplants attached via autotransplantation. The endometriosis model was induced by autotransplantation of endometrium to peritoneal tissue. Peritoneal tissues adjacent to the transplant were obtained at 1, 4, and 7 days posttransplantation. The results showed that messenger RNA expression levels of most of the integrins, collagens, and other ECM reached a peak at 7 days posttransplantation. Uniquely, Lamc2 was significantly increased to its maximum level within 24 hours posttransplantation and may be strongly associated with initiation of the development of endometriosis. These data will be helpful in further investigations of the treatment of endometriosis.