Yuichiro Kudo

Comparative Cytotoxicity Study of Rock Wool and Chrysotile by Cell Magnetometric Evaluation

Rock wool (RW), a type of man-made mineral fiber (MMMF), is a building material used as an asbestos substitute for heat insulation, fire resistance, and reinforcement. RW is included in group 3 of the IARC classification. In the present study, the cytotoxicity of RW was investigated by cell magnetometry, enzyme assay, DNA ladder detection, and electron microscopic morphological evaluation in comparison with chrysotile fibers (CF). Specimens were prepared by 18-h incubation of Fischer rat alveolar macrophages in the presence of RW fibers as the study material, CF as positive control, and phosphate-buffered saline (PBS) as negative control, together with a relaxation indicator, Fe3O4, except for morphological evaluation, followed by additional procedures of external magnetization and subsequent 20-min remanent magnetic field measurement for magnetometric evaluation, and macrophage DNA extraction for evaluating possible apoptosis by DNA ladder detection. In magnetometry, relaxation, a marker of cytotoxicity, was rapid in both the RW- and PBS-treated groups, while it was delayed in both the long and short CF-treated groups. Differences in percent lactate dehydrogenase (LDH) release between the RW-treated group and PBS-treated group were not significant, but those between the RW-treated group and short CF-treated group were statistically significant. A DNA ladder was not detected in any of the study groups. Electron micrographs showed that RW did not cause any change, but CF caused changes in macrophages. Thus, magnetometric measurements suggested no cytotoxicity of RW. We plan, in the future, to evaluate the safety of RW by magnetometric measurement and morphological observation of the lungs in in vivo inhalation experiments.

In vitro toxicity of indium arsenide to alveolar macrophages evaluated by magnetometry, cytochemistry and morphological analysis

The present study was conducted to clarify the toxicity of Indium arsenide (InAs) particles to alveolar macrophages of hamsters by cytomagnetometry, enzyme release assays and morphological examinations. One million alveolar macrophages obtained from hamsters were exposed to 60 microg of ferrosoferric oxide and 2, 4, 10 and 20 microg of InAs particles. Relaxation, which is the rapid decline of strength of the remanent magnetic fields radiating from the alveolar macrophages, was insignificantly delayed and decay constants were not changed due to exposure to such doses of InAs. Because the relaxation is thought to be associated with the cytoskeleton, the exposure to InAs may not have impaired their motor function. An LDH release assay and morphological findings indicate slight damage to macrophages. DNA electrophoresis and the TUNEL method revealed neither necrotic changes nor apoptotic changes. Thus, InAs particles at such doses hardly cause cytostructural changes and cell death.

Behavior of new type of rock wool (HT wool) in lungs after exposure by nasal inhalation in rats

ObjectivesPrevious types of rock wool has been recently replaced with high-temperature wool (HT wool). HT wool is characterized by a chemical composition with a higher concentration of Al2O3 and a lower concentration of SiO2, lower biopersistence, and a higher melting point than previous types of rock wool. To evaluate the safety of HT wool, an asbestos substitute, we examined the biopersistence of HT wool in the lungs, based on changes in fiber count according to the length and fiber size (length and width), by performing a nose-only inhalation exposure study in rats.MethodsMale Fischer 344 rats were exposed to fibers at the target exposure concentration of 30 mg/m3 continuously for 3 hours daily for 5 consecutive days. Rats were sacrificed shortly after exposure, and 1, 2, and 4 weeks after exposure, and their lung tissues were incinerated at a low temperature. Then, fiber counts and sizes in the lungs were analyzed using a phase contrast microscope.ResultsThe fiber count in the lungs 4 weeks after exposure significantly decreased from the baseline value (shortly after exposure). The half-life of fibers calculated from the approximation curve was 34 days for all fibers and 11 days for fibers longer than 20 μm.ConclusionsBoth the length and width significantly decreased 4 weeks after exposure, probably because fibers were ingested by alveolar macrophages, discharged to outside of the body by mucociliary movement, or lysed by body fluid. In future studies, it is necessary to examine the long-term persistence of fibers in the lungs.

Biopersistence of Rock Wool in Lungs after Short-Term Inhalation in Rats

To evaluate the safety of rock wool (RW), an asbestos substitute, we examined the biopersistence of RW fibers in rat lungs based on the changes of fiber number and fiber size (length and diameter) by a nose-only inhalation exposure study. Twenty-four male Fischer 344 rats were exposed to RW fibers at a concentration of 30 mg/m3 continuously for 3 h daily for 5 consecutive days. Six rats each were sacrificed shortly and at 1, 2, and 4 wk after exposure, and their lung tissues were ashed by a low-temperature plasma asher. Then the fiber numbers and fiber sizes in lungs were determined using a phase-contrast microscope and computed image analyzer. During the study period, the arithmetic mean (SD) values of fiber and weight concentrations were 78.5 (35.7) fibers/cm3, and 29.9 (28.3) mg/m3, respectively. The fiber number in lungs 4 wk after exposure significantly decreased from the baseline value (shortly after exposure) (p < .05). The half-life of fibers calculated from the approximate curve was 28 days for all fibers and 16 days for fibers with L > 20 μ m, and the rate of decrease in fiber number was 46.3% at 4 wk after exposure (shortly-after group = 100%). Likewise, both length and diameter significantly decreased at 4 wk after exposure (p < .05), probably because fibers were phagosytosed and digested by alveolar macrophages, discharged to outside of the body by mucociliary movement, or dissolved by body fluid. It will be necessary in the future to further confirm the safety of RW fibers by assessing the biopersistence of fibers in the lungs and their pathological effects in our ongoing study performed in accordance with the guidelines established in the “Methods for Determination of Hazardous Properties for Human Health of Man Made Mineral Fibers” (EC protocol).

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.

Behavior of Rock Wool in Rat Lungs after Exposure by Nasal Inhalation

Behavior of Rock Wool in Rat Lungs after Exposure by Nasal Inhalation: Yuichiro Kudo, et al. Department of Preventive Medicine and Public Health, Kitasato University School of Medicine—To evaluate the safety of rock wool (RW) fibers, we examined the biopersistence of RW fibers in the lungs of rats, based on the changes of fiber number and fiber size in the length and width, in a nose‐only inhalation exposure study. Twenty male Fischer 344 rats (6 to 10 wk old) were exposed to RW fibers at a fiber concentration of 70.6 (20.4) fiber/m3 and a dispersion density of 30.4 (6.6) mg/m3 [arithmetic mean (SD)] continuously for 3 h daily for 5 consecutive days. Five rats each were sacrificed shortly after exposure ended (baseline group) and at 1, 2, and 4 wk after exposure, and their lung tissues were ashed by a low temperature plasma‐asher. The numbers and sizes of fibers in the ash samples were determined using a phase contrast microscope and a computed image analyzer. The fiber numbers in the lungs at 4 wk after exposure had significantly decreased from the baseline value, i. e. shortly after exposure (p<0.05). The half‐lives of RW fibers calculated using the one‐compartment model were 32 d for total fibers and 10 d for fibers longer than 20 μm in length. Fiber number was 53.6% of the baseline at 4 wk after exposure (baseline group=100%). Likewise, fiber sizes had significantly decreased at 4 wk after exposure (p<0.05), probably because fibers had been dissolved in body fluid, phagocytosed by alveolar macrophages or discharged from the body by mucociliary movement. In future studies, it will be necessary to examine the carcinogenicity of RW fibers through long‐term inhalation studies.

Magnetometric Evaluation for Cytotoxicity of Potassium Octatitanate Whisker on Alveolar Macrophages of Fischer 344 Rats

Magnetometric Evaluation for Cytotoxicity of Potassium Octatitanate Whisker on Alveolar Macrophages of Fischer 344 Rats: Mitsuyasu Watanabe, et al. Department of Preventive Medicine and Public Health, Kitasato University School of Medicine—Alveolar macrophages are thought to play a major role in the pathophysiology of lung diseases caused by exposure to various kinds of pathogens and particles. In the present study, the cytotoxic effect of potassium octatitanate whisker (PT) on macrophages was evaluated by means of magnetometry, lactate dehydrogenase (LDH) release measurement, apoptosis measurement and morphological observations. Alveolar macrophages obtained from Fischer rats (F344/N Sic) by bronchoaiveolar lavage were incubated in vitro for 18 h with Fe3O4 as a magnetometric indicator, and PTs as test materials. In the control group and the group exposed to 10 μg/ml of PT, rapid attenuation of the remanent magnetic field (RMF), so called “relaxation,” was observed immediately after cessation of the external magnetic field. In comparison, a delay of relaxation was observed in alveolar macrophages exposed to 20 and 40 μg/ml of PT. The decay constants, which are calculated from decreasing RMF for the first 2 min, in the groups exposed to 20 and 40 μg/ml of PT had significantly lower values than the control. LDH release induced by exposure to 20 and 40 μg/ml of PT increased significantly in a concentration dependent manner in PT‐exposed macrophages, whereas only negligible LDH release was observed in control groups. The level of PT affecting alveolar macrophages was at the same concentration, and in a dose‐dependent manner among relaxation, decay constant and LDH measurement. A DNA ladder detection method and morphological observations detected no apoptosis in PT‐exposed macrophages. Electron microscopic examination revealed vacuolar changes and cell membrane damage in PT‐exposed macrophages, but no significant changes in control macrophages. The results of magnetometry, LDH release, apoptosis measurement and electron microscopic observations suggest concentration dependent cytotoxicity caused by exposure of alveolar macrophages to PT.

Effects of rock wool on the lungs evaluated by magnetometry and biopersistence test

BackgroundAsbestos has been reported to cause pulmonary fibrosis, and its use has been banned all over the world. The related industries are facing an urgent need to develop a safer fibrous substance. Rock wool (RW), a kind of asbestos substitute, is widely used in the construction industry. In order to evaluate the safety of RW, we performed a nose-only inhalation exposure study in rats. After one-month observation period, the potential of RW fibers to cause pulmonary toxicity was evaluated based on lung magnetometry findings, pulmonary biopersistence, and pneumopathology.MethodsUsing the nose-only inhalation exposure system, 6 male Fischer 344 rats (6 to 10 weeks old) were exposed to RW fibers at a target fiber concentration of 100 fibers/cm3 (length [L] > 20 μm) for 6 hours daily, for 5 consecutive days. As a magnetometric indicator, 3 mg of triiron tetraoxide suspended in 0.2 mL of physiological saline was intratracheally administered after RW exposure to these rats and 6 unexposed rats (controls). During one second magnetization in 50 mT external magnetic field, all magnetic particles were aligned, and immediately afterwards the strength of their remanent magnetic field in the rat lungs was measured in both groups. Magnetization and measurement of the decay (relaxation) of this remanent magnetic field was performed over 40 minutes on 1, 3, 14, and 28 days after RW exposure, and reflected cytoskeleton dependent intracellular transport within macrophages in the lung. Similarly, 24 and 12 male Fisher 344-rats were used for biopersistence test and pathologic evaluation, respectively.ResultsIn the lung magnetometric evaluation, biopersistence test and pathological evaluation, the arithmetic mean value of the total fiber concentration was 650.2, 344.7 and 390.7 fibers/cm3, respectively, and 156.6, 93.1 and 95.0 fibers/cm3 for fibers with L > 20 μm, respectively. The lung magnetometric evaluation revealed that impaired relaxation indicating cytoskeletal toxicity did not occur in the RW exposure group. In addition, clearance of the magnetic tracer particles was not significantly affected by the RW exposure. No effects on lung pathology were noted after RW exposure.ConclusionThese findings indicate that RW exposure is unlikely to cause pulmonary toxicity within four weeks period. Lung magnetometry studies involving long-term exposure and observation will be necessary to ensure the safety of RW.

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.

Magnetometric evaluation of toxicities of chemicals to the lungs and cells

Because the lungs are exposed to airborne hazardous materials, alveolar macrophages (AMs) play a major role in defending against the exposure to various noxious chemical substances. In this study, we reviewed magnetometric investigations of the effects of various chemicals on the lungs and AMs. Magnetometry, using magnetite as an indicator, was used to evaluate the effects of certain chemicals on the lung and AMs. A rapid decrease of the remanent magnetic field after the cessation of external magnetization, a phenomenon called relaxation, was impaired when the lungs and macrophages were exposed to toxic substances. The delayed in vivo relaxation observed in the lungs exposed to magnetite and gallium arsenide was almost identical to the in vitro relaxation observed in the AMs exposed to the same materials. Delayed relaxation was observed in the AMs exposed to silica dust; various fibers, such as chrysotile and some man-made mineral fibers; and toxic arsenic and cadmium compounds. The extracellular release of lactate dehydrogenase activity was found in the AMs exposed to the chemicals. Relaxation is attributed to the cytoskeleton-driven rotation of phagosomes containing magnetite. While the exact mechanism of delayed relaxation due to exposure to harmful chemicals remains to be clarified, cell magnetometry appears to be useful for the safety screening of chemical substances.