池上 昭彦

Focusing on the relatively unexplored presence of micro- and nano-plastic aerosol particles, this study quantitatively assessed the emission of nano-plastic particles during the machining of carbon fiber reinforced plastic (CFRP) in the working environment. Measurements of aerosol particles smaller than 1 µm in size were performed by aerosol mass spectrometry. The findings revealed that concentrations of carbonous aerosol particles (organic aerosol and refractory black carbon (rBC)) were higher during working hours than during non-working hours. Positive matrix factorization identified CFRP particles as a significant source, contributing an average of approximately 30% of concentration of carbonous aerosol particles during working hours. This source apportionment was corroborated by the presence of bisphenol A and F fragments, principal components of the epoxy resins used in CFRP, and was corroborated by similarities to the carbon cluster ion distribution observed in rBC during CFRP pipe-cutting operations. Further, the particle size distribution suggested the existence of plastic aerosol particles smaller than 100 nm. This study established the method to quantitatively distinguish nano-plastic aerosol particles from other aerosol particles in high temporal resolution and these techniques are useful for accurately assessing exposure to nano-plastic aerosol particles in working environments.

OBJECTIVES: Carbon fibers are used in a variety of industrial applications, based on their lightweight and high stiffness properties. There is little information on the characteristics and exposure levels of debris generated during the factory processing of carbon fibers or their composites. This study revisits the general assumption that carbon fibers or their debris released during composite processing are considered safe for human health. METHODS: The present interventional study was conducted at a factory located in Japan, and involved on-site collection of debris generated during the industrial processing of polyacrylonitrile (PAN)-based carbon-fiber-reinforced plastic (CFRP). The debris were collected before being exhausted locally from around different factory machines and examined morphologically and quantitatively by scanning electron microscopy. The levels of exposure to respirable carbon fibers at different areas of the factory were also quantified. RESULTS: The collected debris mainly contained the original carbon fibers broken transversely at the fiber's major axis. However, carbon fiber fragments morphologically compatible with the WHO definition of respirable fibers (length: > 5 μm, width: < 3 μm, length/width ratio: > 3:1) were also found. The concentrations of respirable fibers at the six examined factory areas under standard working conditions in the same factory were below the standard limit of 10 fibers/L, specified for asbestos dust-generating facilities under the Air Pollution Control Law in Japan. CONCLUSIONS: Our study identified potentially dangerous respirable fibers with high aspect ratio, which was generated during the processing of PAN-based CFRP. Regular risk assessment of carbon fiber debris is necessary to ensure work environment safety.

BACKGROUND: Allergen-specific immunoglobulins have a crucial role in allergic diseases. Most wheeze episodes develop before school age, and allergic rhinitis later develops during early elementary school years. However, the clinical background and cytokine/chemokine profiles associated with changes in immunoglobulins during early school-age are poorly understood. METHODS: This study used blood samples from children participating in the JECS Pilot Study. We examined nineteen kinds of aeroallergen-specific immunoglobulins (IgE, IgG1, IgG4, and IgA) levels in patients at age 6 and age 8. Fluctuations of Der f 1- and Cry j 1-specific immunoglobulins levels during the two periods were compared to assess the frequency of allergic statuses and clusters of cytokine/chemokine profiles. RESULTS: The medians of aeroallergen-specific IgE levels did not fluctuate, and almost all IgG1 and IgG4 decreased. In IgA, four (e.g., Der f 1) increased, whereas the other four (e.g., Cry j 1) decreased. The ratio of the Der f 1-specific IgG1 level at age 8 to that at age 6 was higher in children with poor asthma control than in children with better asthma control. Moreover, the cytokine/chemokine cluster with relatively lower IL-33 and higher CXCL7/NAP2 was associated with lower Der f 1- and Cry j 1-specific IgG4 levels, but not IgE levels. CONCLUSIONS: The cluster of cytokine/chemokine profiles characterized by lower IL-33 and higher CXCL7/NAP2 was associated with the maintenance of aeroallergen-specific IgG4 levels. This result provides a basis for considering the control of aeroallergen-specific immunoglobulins.

Recent epidemiological studies reported cases of cholangiocarcinoma in workers exposed to 1,2-dichloropropane (1,2-DCP) in an offset proof printing factory in Japan. The present study investigated the effects of 1,2-DCP on the expression of histone family member X (H2AX) phosphorylated on Ser 139 (γ-H2AX), a marker of DNA double strand break, in human immortalized cholangiocytes MMNK-1 cells. Mono-cultures of MMNK-1 cells and co-cultures of MMNK-1 cells with THP-1 macrophages were exposed to 1,2-DCP at concentrations of 100 and 500 μM for 24 h. Expression of γ-H2AX was visualized by immunofluorescence staining. Exposure to 1,2-DCP had no effect on the expression of γ-H2AX in mono-cultured MMNK-1 cells, but significantly increased the number of nuclear foci stained by γ-H2AX in MMNK-1 cells co-cultured with THP-1 macrophages. Exposure to 1,2-DCP also significantly increased the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in co-cultured MMNK-1 cells. The results suggest that macrophages play a critical role by producing cytokines in 1,2-DCP-induced DNA double strand break in MMNK-1 cells.

Objective: Ultra-sensitive hormone assays have detected slight sex differences in blood estradiol (E2) levels in young children before adrenarche. However, the origin of circulating E2 in these individuals remains unknown. This study aimed to clarify how E2 is produced in young girls before adrenarche. Design: This is a satellite project of the Japan Environment and Children's Study organized by the National Institute for Environmental Studies. Methods: We collected blood samples from healthy 6-year-old Japanese children (79 boys and 71 girls). Hormone measurements and data analysis were performed in the National Institute for Environmental Studies and the Medical Support Center of the Japan Environment and Children's Study, respectively. Results: E2 and follicle stimulating hormone (FSH) levels were significantly higher in girls than in boys, while dehydroepiandrosterone sulfate (DHEA-S) and testosterone levels were comparable between the two groups. Girls showed significantly higher E2/testosterone ratios than boys. In children of both sexes, a correlation was observed between E2 and testosterone levels and between testosterone and DHEA-S levels. Moreover, E2 levels were correlated with FSH levels only in girls. Conclusions: The results indicate that in 6-year-old girls, circulating E2 is produced primarily in the ovary from adrenal steroids through FSH-induced aromatase upregulation. This study provides evidence that female-dominant E2 production starts several months or years before adrenarche. The biological significance of E2 biosynthesis in these young children needs to be clarified in future studies.

Prenatal exposure to methylmercury (MeHg) affects child development after birth. However, many epidemiological studies have evaluated total mercury levels without analyzing speciation. Biomonitoring of MeHg and inorganic mercury (IHg) is essential to reveal each exposure level. In this study, we compared a high-throughput analysis for mercury speciation in blood using liquid chromatography-inductively coupled plasma-mass spectrometry (LC-ICP-MS) and liquid chromatography-cold vapor atomic fluorescence spectrometry (LC-CVAFS). The validated LC-ICP-MS method was applied to 101 maternal blood and 366 cord blood samples in the pilot study of the Japan Environment and Children's Study (JECS). The accuracy of the LC-CVAFS method ranged 90-115% determined by reference material analysis. To evaluate the reliability of 366 cord blood samples, fifty cord blood samples were randomly selected and analyzed using LC-CVAFS. The median (5th-95th percentile) concentrations of MeHg and IHg were 5.4 (1.9-15) and 0.33 (0.12-0.86) ng/mL, respectively, in maternal blood, and 6.3 (2.5-15) and 0.21 (0.08-0.49) ng/mL, respectively, in cord blood. Inter-laboratory comparison showed a relatively good agreement between LC-ICP-MS and LC-CVAFS. The median cord blood:maternal blood ratios of MeHg and IHg were 1.3 and 0.5, respectively. By analyzing speciation, we could focus on the health effects of each chemical form.

Fusobacterium nucleatum is a human periodontal pathogen that causes opportunistic infections. It has been implicated in preterm birth and has as a pathogen of colorectal cancer. However, it is a common member of the oral microbiota and can have a symbiotic relationship with its hosts. To date, studies of F. nucleatum have been hindered by a lack of effective genetic tools, and the transformation of F. nucleatum has not been investigated. In this chapter, protocols for the transformation of F. nucleatum strain 12230 using sonoporation are presented. We also include a genetic complementation protocol for a F. nucleatum knockout mutant.

BACKGROUND: The relationship between allergic individuals and their responsiveness to routine vaccines has rarely been investigated. This study examined whether the seroprevalence of measles antibody differed between children with and without allergic diseases in the general pediatric population. METHODS: The cross-sectional study was performed within a prospective general birth cohort (a pilot study of the Japan Environment & Children's Pilot Study [JECS]) of children aged 8 years. The clinical history of allergic diseases, measles, and the concentration of measles immunoglobulin G titers in serum enzyme immunoassay were examined. Fisher's exact tests were used to assess the relationships between the allergic characteristics of the children and their measles antibody positivity rates. RESULTS: This study included 162 children. Any allergic disease was reported in 75 (46.3%). The measles antibody positivity rate was 94.7% among children with any allergic diseases and 92.0% among children without allergic diseases. Our results revealed no differences in measles antibody seropositivity between children with allergies and controls. CONCLUSIONS: Children with allergies mount and maintain a comparable immune response to the measles vaccine.

Lead exposure is associated with impaired neurodevelopment among children. House dust is recognized as one of the important secondary sources of lead exposure in children. We assessed the relationship between lead contamination in house dust and blood lead level in Pakistani children. We investigated lead contamination in house dust samples collected from 59 houses in Karachi, Pakistan. The lead content of house dust in Pakistan was relatively higher than that reported in previous studies. Weekly lead intakes from house dust were considerably higher among Pakistani children. In Pakistani children, 12% (7 of 58) showed lead intake values greater than the previous Provisional Tolerable Weekly Intake of lead. A correlation (Pearson's correlation = 0.37) was found between weekly lead intake from house dust and blood lead level in Pakistani children. In addition, blood lead levels were significantly higher in children with high lead intakes than in children with low and medium lead intakes. Thus, house dust is an important source of lead exposure in Pakistani children.

Objectives Exposure to inorganic arsenic (iAs) is a world-wide health concern. We reported that Japanese children and pregnant women are exposed to moderate levels of iAs through food. Reducing iAs contamination from foods of high iAs is an important issue unique in Japan. Integrated iAs is methylated to less toxic organic forms, and S-adenosyl-L-methyonine (SAM), a common methyl-donor of DNA and histones, is utilized in this process. Chronic consumption of SAM by iAs metabolism due to exposure to iAs might alter the epigenetic modification of genome. The SAM biosynthesis pathway is dependent on folate cycle, and it is possible that ingestion of sufficient folic acid (FA) is protective to iAs induced toxicity. Methods In the course of our cross-sectional body burden analyses of Pb and iAs in Japanese children and pregnant women, termed "PbAs study", FA concentration in serum of 104 pregnant women was measured. Results Mean (±SEM) of serum FA concentration was 15.8 ± 1.3 (ng/mL). There are significant number of people showing very high FA (>30 ng/ mL), and large fraction of them were taking supplements daily. Conclusions These results suggested that level of FA ingestion of Japanese pregnant women is high for supporting normal fetal development.

Nanoparticles (NPs) are widely used in food, and analysis of their potential gastrointestinal toxicity is necessary. The present study was designed to determine the effects of silica dioxide (SiO2), titanium dioxide (TiO2), and zinc oxide (ZnO) NPs on cultured THP-1 monocyte-derived macrophages and human epithelial colorectal adenocarcinoma (Caco-2) cells. Exposure to ZnO NPs for 24 h increased the production of redox response species (ROS) and reduced cell viability in a dose-dependent manner in THP-1 macrophages and Caco-2 cells. Although TiO2 and SiO2 NPs induced oxidative stress, they showed no apparent cytotoxicity against both cell types. The effects of functionalized SiO2 NPs on undifferentiated and differentiated Caco-2 cells were investigated using fluorescently labeled SiO2 NPs with neutral, positive, or negative surface charge. Exposure of both types of cells to the three kinds of SiO2 NPs significantly increased their interaction in a dose-dependent manner. The largest interaction with both types of cells was noted with exposure to more negatively surface-charged SiO2 NPs. Exposure to either positively or negatively, but not neutrally, surface-charged SiO2 NPs increased NO levels in differentiated Caco-2 cells. Exposure of differentiated Caco-2 cells to positively or negatively surface-charged SiO2 NPs also upregulated interleukin-8 expression. We conclude that functionalized surface-charged SiO2 NPs can induce pro-inflammatory responses but are noncytotoxic.

This study aimed to reveal a new dimension of allergy profiles in the general population by using machine learning to explore complex relationships among various cytokines/chemokines and allergic diseases (asthma and atopic dermatitis; AD). We examined the symptoms related to asthma and AD and the plasma levels of 72 cytokines/chemokines obtained from a general population of 161 children at 6 years of age who participated in a pilot birth cohort study of the Japan Environment and Children's Study (JECS). The children whose signs and symptoms fulfilled the criteria of AD, which are mostly based on questionnaire including past symptoms, tended to have higher levels of the two chemokine ligands, CCL17 and CCL27, which are used for diagnosis of AD. On the other hand, another AD-related chemokine CCL22 level in plasma was higher only in children with visible flexural eczema, which is one of AD diagnostic criteria but was judged on the same day of blood examination unlike other criteria. Here, we also developed an innovative method of machine learning for elucidating the complex cytokine/chemokine milieu related to symptoms of allergic diseases by using clustering analysis based on the random forest dissimilarity measure that relies on artificial intelligence (AI) technique. To our surprise, the majority of children showing at least any asthma-related symptoms during the last month were divided by AI into the two clusters, either cluster-2 having elevated levels of IL-33 (related to eosinophil activation) or cluster-3 having elevated levels of CXCL7/NAP2 (related to neutrophil activation), among the total three clusters. Future studies will clarify better approach for allergic diseases by endotype classification.

Poly- and perfluoroalkyl substances (PFAS) have been investigated in a number of cohort studies due to concern over their adverse health effects. The aim of this study was to develop a reliable, high throughput and cost-effective analytical method for a broad range of PFAS in human serum. Protein precipitation, automatic solid phase extraction (SPE) pre-treatment and column-switching LC-MS/MS were employed. The optimised and validated method was then used to analyse the levels of 28 PFAS in 339 maternal serum samples from Pilot Study of the Japan Environment and Children's Study. Perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluoroundecanoic acid (PFUnA) and perfluorooctane sulphonic acid (PFOS) were detected in all 339 samples at median (range) concentrations of 1.9 (0.46-15), 1.5 (0.32-10), 1.3 (0.25-4.5) and 3.7 (0.43-15) ng/ml, respectively. These levels are comparable to those reported in previous studies using samples collected from various parts of the world. With a few exceptions, the remainder of the PFAS examined had lower detection rates but were found at concentrations similar to those reported in previous studies. The sensitivity and throughput ability of the method developed here are sufficient for its application in a large-scale biomonitoring study.

OBJECTIVES: No previous study has used repeated measures data to examine the associations of dog/cat ownership with wheezing and asthma prevalence among children. This prospective study used repeated measurers analysis to determine whether dog/cat ownership in childhood is an independent risk factor for wheezing and asthma, after adjustment for gestational, socio-economical, and demographical confounders confounders, in Japan. METHODS: We conducted a multicenter pilot study of the Japan Environment and Children's Study (JECS) during 2009-2010. Among 440 newborn infants enrolled, 410 (52.8% males) were evaluated for dog/cat ownership in the home and history of wheezing and asthma in five follow-up questionnaire surveys (until age 6 years). Dog/cat ownership during follow-up period was categorized into four groups: 7.6% were long-term dog/cat owners, 5.9% were toddler-age owners, 5.9% were preschool-age owners, and 80.7% were never owners. RESULTS: The prevalence of wheezing during follow-up period increased from 20.8% to 35.4% and the prevalence of asthma increased from 1.3% to 16.3%. A fitted logistic generalized estimating equation models including important confounders showed no significant associations of the interaction between dog and/or cat ownership and follow-up time with the risks of wheezing and asthma. However, the risks of wheezing and asthma were slightly lower for long-term and toddler-age dog/cat owners than for preschool-age and never owners. CONCLUSIONS: The present findings suggest that dog and cat ownership from toddler-age does not increase the risks of wheezing and asthma compared with never owners among Japanese children.