Mitsushi Okada

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.

Magnetometric Evaluation of Cadmium Oxide--Induced Toxicity to Pulmonary Alveolar Macrophages of Syrian Golden Hamsters

Since alveolar macrophages play an important role in the clearance of inhaled dust from airways, these cells have been used as a target for various toxic chemicals. Alveolar macrophages obtained from bronchoalveolar lavage of Syrian golden hamsters were concurrently exposed in vitro to Fe 3 O 4 , as an indicator for magnetometry, and various concentrations of cadmium oxide (CdO) in this study. A rapid decrease of the remnant magnetic field, called relaxation, was observed after the cessation of an external magnetic field stimulus in macrophages concurrently exposed to phosphate-buffered saline or CdO at 0.1 w g/ml, while relaxation was delayed in those concurrently exposed to 1, 25, or 50 w g/ml CdO. Therefore, the concentration of CdO affecting relaxation in vitro was estimated at between 0.1 and 1 w g/ml. Release of LDH activity from CdO-exposed macrophages into the medium significantly increased at levels of 25 and 50 w g/ml CdO. Apoptosis was not detected in macrophages exposed to CdO by the DNA ladder detection method or morphological observations. Electron-microscopic examination revealed severe membrane damage and vacuolar changes in macrophages exposed to CdO. Since delayed relaxation is thought to occur by (1) disrupted cytoskeleton-driven random rotation of phagosomes containing iron oxide particles, (2) significant lactate dehydrogenase (LDH) activity release, and (3) detachment of cell membranes, CdO is considered to affect macrophage functions.

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.

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.