Masashi Tsunoda

Tributyltin (TBT) increases TNFα mRNA expression and induces apoptosis in the murine macrophage cell line in vitro

ObjectiveTributyltin (TBT) compounds have been widely used as antifouling agents for shipbottom paint. The immune system is a target of TBT intoxication. We evaluated the effects of TBT chloride in macrophages, which have critical roles in the immune system, using a murine macrophage lineage cell line, J774.1,in vitro.MethodsWe examined tumor necrosis factor α (TNFα), interleukin-1β (IL-1β) andc-jun mRNA expression in J774.1 cells. The effects of TBT on the apoptosis of J774.1 cells were examined by determining the percentage of TUNEL-positive cells and caspase-3 activity.ResultsThe mean values of the viabilities of J774.1 cells exposed to TBT decreased dose-dependently. The relative mRNA expression of TNFα increased dose-dependently, however, that of IL-1β was not significantly different among the groups. The mean percentage of TUNEL-positive cells increased dose-dependently. Increases in the caspase-3 activities of J774.1 cells were observed in the groups exposed to higher concentrations of TBT. The mean value of relative mRNA expression of c-Jun transcription factor increased dose-dependently.ConclusionsThe increases in the percentage of TUNEL-positive cells and in caspase-3 activity suggested that exposure to TBT induces apoptosis of J774.1 cells. The increases in the mRNA expression of TNFα andc-jun by TBT may be related to apoptosis in macrophages.

Effect of tributyltin compound onN-methyl-D-aspartate (NMDA) receptors in brain of preweanling mice

ObjectiveThe aim of this study was to investigate the effect of tributyltin (TBT) compound onN-methyl-d-aspartate (NMDA) receptors in the brains of preweanling mice.MethodsPregnant ICR mice were exposed to TBT chloride at concentrations of 0, 15, and 50 ppm in water. Male offspring were sacrificed at 1, 2 and 3 weeks after birth. Mouse brain membranes were prepared from cerebral cortices, and the specific binding of [3H]MK-801 to an NMDA receptor was determined by radioligand binding assay.ResultsThe mean body weight of preweanling mice of the 50 ppm dose group decreased by 17–25% (p<0.01) at 1, 2 and 3 weeks of age, compared with that of preweanling mice of the corresponding control group. The [3H]MK-801 binding level significantly decreased (p<0.05) in the 15 ppm F1 group at 1 week and in the 15 ppm and 50 ppm F1 groups at 3 weeks of age, compared with that in the corresponding control F1 group.ConclusionsThe exposure to TBT via placenta and dams milk seriously affected not only the growth of preweanling mice, but also the F1 cerebral NMDA receptors involved in memory and learning.

The increases in relative mRNA expressions of inflammatory cytokines and chemokines in splenic macrophages from rats exposed to multi-walled carbon nanotubes by whole-body inhalation for 13 weeks

Abstract Background: The toxicity of multi-walled carbon nanotubes (MWCNT) may be related to the immune system. The objective of this study was to obtain information for immunotoxic mechanisms of MWCNT in situ. Methods: Using whole-body inhalation, male and female rats were exposed to 0, 0.2, 1 or 5 mg MWCNT/m3 for 13 weeks. Thereafter, spleens were recovered from the rats. Real-time PCR was done to assess expression of TNFα, IL-1β, IL-6, IL-10, MCP-1 and MIP-1α mRNA in the splenic macrophages; splenic T-lymphocytes were examined for IL-2 and TGF-β1 mRNA expression. Results: The relative expression of IL-1β mRNA in the cells from female rats exposed to 5 mg MWCNT/m3 was significantly higher than that in control cells. For IL-6 and IL-10, cells from rats in the 0.2 and 5 mg MWCNT/m3 had significantly higher mRNA expressions than did cells from controls. Expression of IL-1β, IL-6 and TNFα genes in cells from males in all exposure groups were higher than in control cells. Expression of MIP-1α in the cells from female 5-mg group was significantly higher than that in cells in the control. Only IL-2 was expression reduced, i.e. cells from male and female rats in all MWCNT groups had significantly lower mRNA expressions than control cells. Conclusions: Systemic inflammation would likely occur in rats (or other hosts) exposed to MWCNT via inhalation due to increases in the expression of inflammatory cytokines in splenic macrophages. Moreover, decreases in IL-2 expression in T-lymphocytes may be critical to the potential reductions in anti-tumor responses in MWCNT-exposed hosts.

The Cytotoxicity of Microglass Fibers on Alveolar Macrophages of Fischer 344 Rats Evaluated by Cell Magnetometry, Cytochemisry and Morphology

ObjectivesThe toxicity of microglass fibers (MG), one of the man-made mineral fibers, has not been sufficiently evaluated. The aim of the current study was to evaluate the cytotoxicity of MGin vitro.MethodsAlveolar macrophages were obtained from the bronchoalveolar lavage of male F344/N rats. The macrophages were exposed to MG at concentrations of 0, 40, 80, 160 and 320 μg/ml. The effects of MG on the macrophages were examined by cell magnetometry, LDH assay and morphological observation.ResultsIn the cell magnetometry experiment, a significant delay of relaxation (the reduction of remanent magnetic field strength) was observed in the cells treated with 160 and 320 μg/ml of MG in a dose-dependent manner. A significant increase in LDH release was also observed in the cells with 160 and 320 μg/ml in a dose-dependent manner. Changes in the cytoskeleton were observed after exposure to MG by immunofluorescent microscopy using an α-tubulin antibody.ConclusionsThe cytotoxicity of MG on alveolar macrophages was demonstrated with cell magnetometry. The mechanism of the toxic effects of MG was related to cytoskeleton damage.

Fluoride in drinking water exacerbates glomerulonephritis and induces liver damage in ICR-derived glomerulonephritis mice

To evaluate the effects of fluoride on the kidney and the liver of ICR-derived glomerulonephritis (ICGN) mice by using laboratory tests and pathological examinations, fluoride was administered to the ICGN mice at 0, 25, 50, 100, and 150 ppm in drinking water for 4 weeks and to the ICR mice, which have normal kidney function at 0 and 150 ppm. The BUN, creatinine, GOT, and GPT in the serum of each mouse were determined. When a mouse died, the sample from the day closest to the death was assigned for the mean. Pathological changes in the kidney were examined after PAS (periodic acid-Schiff) staining. All of the ICGN mice in the 150 ppm group and one of seven in the 100 ppm group died before the end of week 4, but no ICR mice died. For ICGN mice, the mean value of body weight in the 150 ppm group was significantly lower than those in 0 ppm group and other fluoride-administered groups. The mean values of relative liver and kidney weights in the 100 and 150 ppm groups were significantly lower than those in the control. The mean values of BUN, creatinine, and GPT in the 150 ppm group were significantly higher than those in the control. The thickness of the glomerular capillary wall and the increased mesangial matrix in the kidney were prominent in the fluoride-administered ICGN mice. These results suggested that fluoride severely exacerbated glomerulonephritis and tublar-intestitial changes in ICGN mice.

Fluoride potentiates tubulointerstitial nephropathy caused by unilateral ureteral obstruction

The contamination of ground water by fluoride has been reported worldwide. Most fluoride (approximately 70%) is filtered by the kidneys; humans or experimental animals with renal damage therefore may be more affected by fluoride exposure than those with normal kidney function. Tubulointerstitial fibrosis, which involves macrophage-promoted extracellular matrix production and myofibroblast migration, can be induced in rats by unilateral ureteral obstruction (UUO). We examined the effects of fluoride exposure on tubulointerstitial fibrosis in the obstructed kidney of UUO rats. The left ureters of 6-week-old male rats were ligated using silk sutures. Fluoride was then administered for 2 weeks at doses of 0, 75, and 150ppm in the drinking water. Real-time polymerase chain reaction was performed to analyze transforming growth factor beta 1 (TGF-β1) transcription; histological and immunohistochemical staining were used to identify positive areas within the renal cortex and staining-positive cells by image analysis. Significant increases were observed in the obstructed kidneys of UUO rats exposed to 150ppm fluoride (compared to 0ppm) for areas or number of cells that stained with Masson trichrome or with antibodies against collagen type I, alpha-smooth muscle actin (α-SMA, a myofibroblast marker), ED1, ED2, and ED3 (macrophage markers), and TGF-β1. Taken together, these observations suggested that fluoride exacerbates tuburointerstitial nephropathy resulting from UUO, and that this effect occurs via activation of the M2 macrophage-TGF-β1-fibroblast/myofibroblast-collagen synthesis pathway.

Occupational Exposure Limits of lead, dimethylamine, n-butyl-2,3-epoxypropyl ether, and 2-ethyl-1-hexanol and carcinogenicity and occupational sensitizer classification.

The Committee for Recommendation of Occupational Exposure Limits, Japan Society for Occupational Health Kenichi Azuma, Ginji Endo, Yoko Endo, Kunio Hara, Koji Harada, Hajime Hori, Seichi Horie, Hyogo Horiguchi, Masayoshi Ichiba, Gaku Ichihara, Masayuki Ikeda, Tatsuya Ishitake, Akiyoshi Ito, Satoko Iwasawa, Michihiro Kamijima, Kanae Karita, Toshio Kawai, Toshihiro Kawamoto, Akio Koizumi, Shinji Kumagai, Yukinori Kusaka, Muneyuki Miyagawa, Yasuo Morimoto, Kasuke Nagano, Tamie Nasu, Tetsuo Nomiyama, Kazuyuki Omae, Kazuhiro Sato, Hirokazu Okuda, Haruhiko Sakurai, Tomotaka Sobue, Yasushi Suwazono, Toru Takebayashi, Tatsuya Takeshita, Akito Takeuchi, Masatoshi Tanaka, Shigeru Tanaka, Teruomi Tsukahara, Masashi Tsunoda, Susumu Ueno, Yuko Yamano, Takenori Yamauchi and Eiji Yano

Skin irritation to glass wool or continuous glass filaments as observed by a patch test among human Japanese volunteers.

Glass wool and continuous glass filaments have been used in industry. We examined the irritability of those among Japanese. A patch test was performed on 43 volunteers for the followings: glass wool for non-residential use with and without a urea-modified phenolic resin binder, that for residential use with and without the binder, and continuous glass filaments with diameters of 4, 7, 9, and 13 µm. Materials were applied to an upper arm of each volunteer for 24 h. The skin was observed at 1 and 24 h after the removal. At 1 h after removal, slight erythema was observed on the skin of a woman after the exposure to glass wool for residential use without the binder. Erythema was observed on the skin of another woman at 1 h after a 24-h exposure to glass wool for non-residential use without the binder. There were no reactions at 24 h after the removal. The low reactions in the patch test suggested that the irritability caused by glass wool, irrespective of a resin component, could be induced mechanically, and that the irritability caused by continuous glass filaments with resin could be slight and either mechanical or chemical.

Occupational exposure limits for ethylene glycol monobutyl ether, isoprene, isopropyl acetate and propyleneimine, and classifications on carcinogenicity, occupational sensitizer and reproductive toxicant

The Committee for Recommendation of Occupational Exposure Limits, Japan Society for Occupational Health Kenichi Azuma, Ginji Endo, Yoko Endo, Tetsuhito Fukushima, Kunio Hara, Hajime Hori, Seichi Horie, Hyogo Horiguchi, Masayoshi Ichiba, Gaku Ichihara, Masayuki Ikeda, Tatsuya Ishitake, Akiyoshi Ito, Yuki Ito, Satoko Iwasawa, Michihiro Kamijima, Kanae Karita, Takahiko Katoh, Toshio Kawai, Toshihiro Kawamoto, Reiko Kishi, Shinji Kumagai, Yukinori Kusaka, Akiko Matsumoto, Muneyuki Miyagawa, Hiroyuki Miyauchi, Yasuo Morimoto, Kasuke Nagano, Hisao Naito, Tamie Nakajima, Tetsuo Nomiyama, Hirokazu Okuda, Kazuyuki Omae, Haruhiko Sakurai, Kazuhiro Sato, Tomotaka Sobue, Yasushi Suwazono, Toru Takebayashi, Tatsuya Takeshita, Akito Takeuchi, Ayano Takeuchi, Masatoshi Tanaka, Shigeru Tanaka, Teruomi Tsukahara, Masashi Tsunoda, Susumu Ueno, Jun Ueyama, Yumi Umeda, Yuko Yamano, Takenori Yamauchi and Eiji Yano

Safety Evaluation of Self-assembling Peptide Gel after Intracranial Administration to Rats Using the Open Field Test

Self-assembling peptides have been developed as clinical materials, which could scaffold to regenerate nerve cells and hemostatic materials in vivo. However, there has not been enough information for their in vivo application. The safety of self-assembling peptides for the application on the brain was examined using behavioral tests for each rat in this study. Self-assembling peptide gel was administered to the surface of the brain at a volume of 20 µL at 1.5%. After 2 months, the open field test and the prepulse inhibition (PPI) test were performed. There were no significant differences between the peptide gel and the control groups in locomotor distances and in %PPIs in the PPI test. The mean values of the percentage of time the rats stayed in the central area of the open field during the first 5 min and instances of center rearing or face washing in the peptide gel group were significantly higher than those in the control. There were amorphous substance in the subarachnoid region, and infiltrations of mononuclear cells were also observed in the self-assembling peptide gel group. Although the meaning of the effects observed in this study was not fully elucidated, the self-assembling gel produced marginal but significant behavioral and histological effects.