Yuji Fujitani

Uptake and cytotoxic effects of multi-walled carbon nanotubes in human bronchial epithelial cells

Carbon nanotubes (CNT) are cytotoxic to several cell types. However, the mechanism of CNT toxicity has not been fully studied, and dosimetric analyses of CNT in the cell culture system are lacking. Here, we describe a novel, high throughput method to measure cellular uptake of CNT using turbimetry. BEAS-2B, a human bronchial epithelial cell line, was used to investigate cellular uptake, cytotoxicity, and inflammatory effects of multi-walled CNT (MWCNT). The cytotoxicity of MWCNT was higher than that of crocidolite asbestos in BEAS-2B cells. The IC(50) of MWCNT was 12 μg/ml, whereas that of asbestos (crocidolite) was 678 μg/ml. Over the course of 5 to 8 h, BEAS-2B cells took up 17-18% of the MWCNT when they were added to the culture medium at a concentration of 10 μg/ml. BEAS-2B cells were exposed to 2, 5, or 10 μg/ml of MWCNT, and total RNA was extracted for cytokine cDNA primer array assays. The culture supernatant was collected for cytokine antibody array assays. Cytokines IL-6 and IL-8 increased in a dose dependent manner at both the mRNA and protein levels. Migration inhibitory factor (MIF) also increased in the culture supernatant in response to MWCNT. A phosphokinase array study using lysates from BEAS-2B cells exposed to MWCNT indicated that phosphorylation of p38, ERK1, and HSP27 increased significantly in response to MWCNT. Results from a reporter gene assays using the NF-κB or AP-1 promoter linked to the luciferase gene in transiently transfected CHO-KI cells revealed that NF-κB was activated following MWCNT exposure, while AP-1 was not changed. Collectively, MWCNT activated NF-κB, enhanced phosphorylation of MAP kinase pathway components, and increased production of proinflammatory cytokines in human bronchial epithelial cells.

Measurement of the Physical Properties of Aerosols in a Fullerene Factory for Inhalation Exposure Assessment

Assessment of human exposure is important for the elucidation of potential health risks. However, there is little information available on particle number concentrations and number size distributions, including those of nanoparticles, in the working environments of factories producing engineered nanomaterials. The authors used a scanning mobility particle sizer and an optical particle counter to measure the particle number size distributions of particles ranging in diameter (D p ) from 10 nm to >5000 nm in a fullerene factory and used scanning electron microscopy to examine the morphology of the particles. Comparisons of particle size distributions and morphology during non-work periods, during work periods, during an agitation process, and in the nearby outdoor air were conducted to identify the sources of the particles and to determine their physical properties. A modal diameter of 25 nm was found in the working area during the non-work period; this result was probably influenced by ingress of outdoor air. During the removal of fullerenes from a storage tank for bagging and/or weighing, the particle number concentration at D p <50 nm was no greater than that in the non-work period, but the concentration at D p >1000 nm was greater during the non-work period. When a vacuum cleaner was in use, the particle number concentration at D p <50 nm was greater than that during the non-work period, but the concentration at D p >1000 nm was no greater. Scanning electron microscopy revealed that the coarse particles emitted during bagging and/or weighing were aggregates/agglomerates of fullerenes; although origin of particles with D p <50 nm is unclear.

Spatial learning and memory function-related gene expression in the hippocampus of mouse exposed to nanoparticle-rich diesel exhaust.

Diesel exhaust particles are a major constituent of ambient particulate matter, and most particles emitted directly from diesel exhaust are smaller than 1microm in diameter. Recently, the toxicity of diesel engine-derived nanoparticles has come to be recognized as an emerging social issue. In the present study, we investigated spatial learning ability and memory function-related gene expressions in mouse hippocampus after the exposure of animals to nanoparticle-rich diesel exhaust (NRDE) with or without a bacterial cell wall component. Lipoteichoic acid (LTA), a cell wall component derived from Staphylococcus aureus, was used to induce systemic inflammation. Male BALB/c mice were exposed to clean air (particle concentration, 4.58microg/m(3)) or NRDE (148.86microg/m(3)) for 5h per day on 5 days of the week for 4 weeks in an exposure chamber, with or without the weekly intraperitoneal injection of LTA. On the day after the final day of exposure, we used a Morris water maze apparatus to examine the ability of the animals to perform a spatial learning task. After the completion of the test, the animals were sacrificed and the hippocampus was collected from each mouse; the expressions of NMDA receptor subunits (NR1, NR2A and NR2B), proinflammatory cytokines (IL-1beta and TNF-alpha) and the oxidative stress marker heme oxygenase 1 were then investigated using real-time RT-PCR. In the Morris water maze task, NRDE/LTA (+) group took a longer time to reach the hidden platform than clear air/LTA (-) group. However, NRDE exposure alone did not affect it. The relative mRNA levels of the NMDA receptor subunits and proinflammatory cytokines were higher in hippocampus of NRDE/LTA (+) group compared to clear air/LTA (-) group. These results indicate that co-exposure of NRDE and LTA could affect spatial learning and memory function-related gene expressions in mouse hippocampus.

Effects of in utero exposure to nanoparticle-rich diesel exhaust on testicular function in immature male rats.

We investigated the effects of in utero exposure to nanoparticle-rich diesel exhaust (NR-DE) on reproductive function in male rats. Pregnant F344 rats were exposed to NR-DE (148.86 microg/m(3), 1.83 x 10(6)particles/cm(3), 3.40 ppm CO, 1.46 ppm NOx), filtered diesel exhaust (F-DE; 3.10 microg/m(3), 2.66 particles/cm(3), 3.30 ppm CO, 1.41 ppm NOx), or clean air (as a control) from gestation days 1 to 19 (gestation day 0=day of sperm-positivity). Male offspring were examined on postnatal day 28. The relative weights of the seminal vesicle and prostate to body weight were decreased after exposure to NR-DE or F-DE compared with controls. Serum concentrations of testosterone, progesterone, corticosterone, and follicle stimulating hormone and testicular concentrations of steroidogenic acute regulatory protein and 17beta-hydroxysteroid dehydrogenase mRNA were decreased after exposure to NR-DE or F-DE compared with control levels. In contrast, serum concentrations of immunoreative inhibin were increased after exposure to NR-DE or F-DE compared with control levels, whereas transcription of follicle stimulating hormone receptor mRNA was increased in the NR-DE exposure group only. These results suggest that prenatal exposure to NR-DE or F-DE leads to endocrine disruption after birth and suppresses testicular function in male rats. Because both the NR-DE and F-DE-exposed groups reacted to the same extent, the nanoparticles in DE did not contribute to the observed reproductive toxicity.

Nanoparticle-rich diesel exhaust affects hippocampal-dependent spatial learning and NMDA receptor subunit expression in female mice

Abstract We investigated the effect of exposure to nanoparticle-rich diesel exhaust (NRDE) on hippocampal-dependent spatial learning and memory function-related gene expressions in female mice. Female BALB/c mice were exposed to clean air, middle-dose NRDE (M-NRDE), high-dose NRDE (H-NRDE) or filtered diesel exhaust (F-DE) for three months. A Morris water maze apparatus was used to examine spatial learning. The expression levels of the N-methyl-D-aspartate (NMDA) receptor subunit, proinflammatory cytokines and neurotrophin mRNAs in the hippocampus were then investigated using real-time RT-PCR. Mice exposed to H-NRDE required a longer time to reach the hidden platform and showed higher mRNA expression levels of the NMDA receptor subunit NR2A, the proinflammatory cytokine CCL3, and brain-derived neurotrophic factor (BDNF) in the hippocampus, compared with the findings in the control group. These results indicate that three months of exposure to NRDE affected spatial learning and memory function-related gene expressions in the female mouse hippocampus.

Effects of inhaled nanoparticle-rich diesel exhaust on regulation of testicular function in adult male rats

We investigated the effects of nanoparticle-rich diesel exhaust (NR-DE) on reproductive function. Eight-week-old male F344 rats were divided into 12 experimental groups and exposed to either whole NR-DE at low (15.37 μg/m3, 2.27 × 105 particles/cm3), middle (36.35 μg/m3, 5.11 × 105 particles/cm3), or high (168.84 μg/m3, 1.36 × 106 particles/cm3) concentrations or clean air for 4, 8, or 12 weeks (5 hours/day, 5 days/week). NR-DE exposure for 4 or 8 weeks did not affect body weight; however, body weight was significantly decreased in rats exposed to low- or high- concentration NR-DE for 12 weeks compared to the control group. Relative weights of testes, epididymides, seminal vesicles, and prostate had increased non-significantly in all NR-DE-exposed rats at 4, 8, and 12 weeks. Adrenal gland relative weights were significantly increased at 4 weeks in rats exposed to low-concentration NR-DE. Plasma luteinizing hormone and follicle stimulating hormone concentrations did not change significantly. Plasma testosterone concentrations were significantly increased after exposure to low- or middle-concentration NR-DE for 4 or 8 weeks compared to controls. Plasma immunoreactive (ir-) inhibin concentrations were significantly increased after exposure to high-concentration NR-DE for 4 weeks or middle- or high-concentration NR-DE for 12 weeks compared to controls. Testicular testosterone concentrations were significantly increased at 4, 8, and 12 weeks after exposure to low-concentration NR-DE compared to controls. In contrast, with exposure to low- or high-concentration NR-DE, testicular ir-inhibin concentrations were significantly greater than in controls, but only at 4 weeks. These results suggest that NR-DE inhalation disrupts the endocrine activity of the male reproductive system.

Clarification of the predominant emission sources of antimony in airborne particulate matter and estimation of their effects on the atmosphere in Japan

Environmental context. The remarkable enrichment of potentially toxic antimony (Sb) in inhalable airborne particulate matter has become of great environmental concern among recent air pollution issues. The present study clarifies the predominant sources of Sb by focusing on the similarities in elemental composition, particle size distributions, and microscopic images found in ambient airborne particles and several potential sources. We identify automotive brake abrasion dust and fly ash emitted from waste incineration as dominant sources of atmospheric Sb in Japan. These results will contribute towards an in-depth understanding of the cycles and fates of Sb in the environment. Abstract. By focusing on the similarities in elemental composition, particle size distributions of elemental concentrations, and microscopic images between ambient airborne particulate matter (APM) and several potential sources, we discuss the predominant sources of antimony (Sb) in APM in Japan. The distribution of Sb concentration in size-classified ambient APM showed a characteristic bimodal profile in which peaks were found in coarse (3.6–5.2 μm) and fine (0.5–0.7 μm) fractions. Elemental ratios, particle sizes, and microscopic images observed in the coarse APM fractions were found to be in good agreement with those of brake abrasion dust. However, in the fine APM fractions, fly ash originating from waste incineration was identified as the most probable source of Sb. Chemical mass balance analysis was performed to determine the effects of the emission sources of Sb, and it was revealed that brake abrasion dust and waste fly ash were the dominant sources of Sb in the coarser and the finer fractions of APM, respectively. The present study provides important clues to understanding the cycles and fates of Sb in the environment.

Seasonal differences of the atmospheric particle size distribution in a metropolitan area in Japan

We compared the effect of ambient temperature observed in two different seasons on the size distribution and particle number concentration (PNC) as a function of distance (up to ~250 m) from a major traffic road (25% of the vehicles are heavy-duty diesel vehicles). The modal particle diameter was found between 10 and 30 nm at the roadside in the winter. However, there was no peak for this size range in the summer, even at the roadside. Ambient temperature affects both the atmospheric dilution ratio (DR) and the evaporation rate of particles, thus it affects the decay rate of PNC. We corrected the DR effect in order to focus on the effect of particle evaporation on PNC decay. The decay rate of PNC with DR was found to depend on the season and particle diameter. During the winter, the decay rate for smaller particles (<30 nm) was much higher (i.e., the concentration decreased significantly against DR), whereas it was low during the summer. In contrast, for particles >30 nm in diameter, the decay rate was nearly the same during both seasons. This distinction between particles less than or greater than 30 nm in diameter reflects differences in particle volatility properties. Mass-transfer theory was used to estimate evaporation rates of C20-C36 n-alkane particles, which are the major n-alkanes in diesel exhaust particles. The C20-C28 n-alkanes of 30-nm particles completely evaporate at 31.2 °C (summer), and their lifetime is shorter than the transport time of air masses in our region of interest. Absence of the peak at 10-30 nm and the low decay rate of PNC <30 nm in diameter in the summer were likely due to the evaporation of compounds of similar volatilities comparable to the C20-C36 n-alkanes from particles near the exhaust pipes of vehicles, and complete evaporation of semivolatile materials before they reached the roadside. These results suggest that the lifetime of particles <30 nm in diameter depends on the ambient temperature, which differs between seasons. This leads us to conclude that these particles show distinctly different spatial distributions depending on the season.

Characterization of Dilution Conditions for Diesel Nanoparticle Inhalation Studies

Diesel exhaust nanoparticles easily coagulate during transportation from the engine to the inhalation chamber, depending on concentrations and residence times. Although dilution is effective in suppressing coagulation growth of nanoparticles, volatile organic carbon (OC) evaporates as a result of dilution. Thus, the design of an inhalation facility to investigate the health effects of nanoparticle-rich exhaust is important. In this study, we determined the optimum dilution conditions in consideration of coagulation growth and evaporation of OC for inhalation studies of nanoparticle-rich diesel exhaust. We found that a short residence time prevented coagulation growth in the primary dilution tunnel after the primary dilution or before the diluted exhaust reached the inhalation chamber after the secondary dilution. However, due to the longer residence time in the inhalation chamber, the coagulation growth occurred in the inhalation chamber depending on secondary dilution ratio which controlled exposure dose (particle concentration in the inhalation chamber). We determined that the secondary dilution ratio for the high-concentration chamber should be around 4.5 times to prevent coagulation growth and to obtain the desired exposure dose. We also found that the loss of OC was relatively independent of the secondary dilution ratio when the secondary dilution ratio was more than 10 times because it seemed to reach a gas–particle equilibrium in the inhalation chamber. We therefore set the secondary dilution ratios for the middle- and low-concentration chambers to 13.5 and 40.5 times, respectively.

Thermal desorption - comprehensive two-dimensional gas chromatography coupled with tandem mass spectrometry for determination of trace polycyclic aromatic hydrocarbons and their derivatives.

We developed a highly sensitive method for determination of polycyclic aromatic hydrocarbons (PAHs) and their derivatives (oxygenated, nitrated, and methylated PAHs) in trace particulate samples by using thermal desorption followed by comprehensive two-dimensional gas chromatography coupled with tandem mass spectrometry (TD-GC×GC-MS/MS) with a selected reaction monitoring mode. The sensitivity of TD-GC×GC-MS/MS was greater than that of TD-GC-HRMS and TD-GC×GC-QMS by one or two orders of magnitude. The instrumental detection limits were 0.03-0.3pg (PAHs), 0.04-0.2pg (oxygenated PAHs), 0.03-0.1pg (nitrated PAHs), and 0.01-0.08pg (methylated PAHs). For small amounts (10-20μg) of standard reference materials (SRMs 1649a and 1650b, urban dust and diesel exhaust particles, respectively), the values measured by using TD-GC×GC-MS/MS agreed with the certified or reference values within a factor of two. Major analytes were quantified successfully by TD-GC×GC-MS/MS from diesel exhaust nanoparticles (18-32nm) and accumulation-mode particles (100-180nm) from an 8-L diesel engine with no exhaust after-treatment system. The PAH profiles differed among driving conditions but they did not differ markedly among the particle sizes.