Cytotoxicity study of rock wool by cell magnetometric evaluation.

The cytotoxicity of rock wool (RW), an asbestos substitute, was evaluated by cell magnetometry. Alveolar macrophages were isolated from male Fisher rats. Following addition of triiron tetraoxide (Fe(3)O(4)) to macrophages, RW was added. Then, the remnant magnetic field strength was measured for 20min after magnetization by an external field. Relaxation, an indicator of decay of cytotoxicity, was observed by cell magnetometry immediately postmagnetization in the group to which RW was added. In general, materials phagocytosed by macrophages are ingested into phagosomes and digested while migrating. This migration of phagosomes occurs by polymerization and depolymerization of the cytoskeleton. As a result of evaluation, relaxation was not delayed by addition of RW, since RW caused no effect on the cytoskeleton. It was suggested that RW has no cytotoxicity as evaluated by cell magnetometry.

Multiplicative correction of subject effect as preprocessing for analysis of variance.

The procedure of repeated-measures ANOVA assumes the linear model in which effects of both subjects and experimental conditions are additive. However, in electroencephalography and magnetoencephalography, there may be situations where subject effects should be considered to be multiplicative in amplitude. We propose a simple method to normalize such data by multiplying each subject's response by a subject-specific constant. This paper derives ANOVA tables for such normalized data. Present simulations show that this method performs ANOVA effectively including multiple comparisons provided that the data follows the multiplicative model.

Fusion and molecular aspects of liposomal nanocarriers incorporated with isoprenoids.

The present study was conducted to investigate whether typical isoprenyl compounds (TICs) can control liposomal fusion reactions through changes in the physical properties of membranes. The fusion capabilities of TIC-incorporated liposomes were characterized by measuring the 13C spin-lattice relaxation times (13CT1) and the gel permeation chromatogram (GPC) patterns. The 13CT1 relaxivities of some of these TIC-liposomes were remarkably enhanced at 27 degrees C. The highest 13CT1 value obtained was for the beta-carotene-liposome, which ruptured, and was attributed to the highest membrane fusion reactivity. The other TIC-liposomes incorporated with alpha-tocopherol, canthaxanthin, or coenzyme Q10 also induced significant fusion and did not rupture in comparison with the beta-carotene-liposome. These results show that the incorporations of TICs into lipid bilayers are useful to control liposomal nanocarriers for suitable membrane packing and advantageous phase separation, which could affect membrane-related processes.

Magnetometric evaluation of the effects of man-made mineral fibers on the function of macrophages using the macrophage cell line RAW 264.7.

The toxic effects of man-made mineral fibers (MMMFs) have been evaluated by cell magnetometry using alveolar macrophages (AMs). Recently, on the other hand, the murine macrophage cell line, RAW 264.7, became available and has been used as an in vitro model of AMs. The objective of this study was to determine whether or not cell magnetometry using RAW 264.7 cells can be used to evaluate the toxic effects of MMMFs. RAW 264.7 cells were exposed to one of the MMMFs, potassium octatitanate (PT) or silicon carbide whisker (SiC) at 0, 20, 40 and 60 microg/ml, or chrysotile as a positive control at 0, 15, 20 and 25 microg/ml. The toxic effects of fibers were evaluated by cell magnetometry and LDH assay. For this comparison, AMs were also exposed to chrysotile fibers (CF). In the RAW 264.7 cells exposed to PT 20, 40, 60 or SiC 20, 40, 60, CF 15, 20 and 25 microg/ml, significant delayed relaxation were observed compared with the respective control. In the LDH assay, significant increases in LDH in the supernatant of the cells exposed to PT 20, 40, 60, SiC 20, 40, 60 and CF 15, 20, 25 microg/ml were observed. In AMs exposed to CF 20, 25 microg/ml significant delayed relaxation and significant increases in LDH compared with the control were observed. The levels of MMMFs that induced significant differences were similar for cell magnetometry and LDH. The levels of CF that induced significant differences in cell magnetometry and LDH were identical for RAW 264.7 cells and AMs. Our results suggest that cell magnetometry using RAW 264.7 cells is adequate to evaluate the cytotoxicity of exposure to MMMFs.

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.

Differences in the effects of fibrous and particulate titanium dioxide on alveolar macrophages of Fischer 344 rats.

Alveolar macrophages are considered to play a major role in the pathophysiology of lung diseases caused by exposure to various kinds of pathogens and particles. In this study, the cytotoxic effect of different shapes of titanium dioxide (TiO(2)) was evaluated on macrophages using a unique magnetometry method and was compared with conventional methods of lactate dehydrogenase (LDH) release, apoptosis measurement, and morphological observations. Alveolar macrophages obtained from Fischer rats (F344) by bronchoalveolar lavage were incubated in vitro for 18 h with Fe(3)O(4) as a magnetometric indicator and fibrous and particulate forms of TiO(2) as test materials. In the control and particulate exposed group, rapid attenuation of the residual magnetic field, 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 fibrous TiO(2). LDH released into serum-free medium induced by exposure to TiO(2) increased significantly in a concentration-dependent manner in macrophages exposed to fibrous TiO(2), while negligible LDH release was observed in macrophages exposed to particulate TiO(2). The DNA ladder detection method and morphological examination detected no apoptosis in macrophages exposed to 60 micro g/ml of fibrous or particulate TiO(2). Electron microscopic examination revealed vacuolar changes and cell surface damage in macrophages exposed to fibrous TiO(2), but no significant changes in macrophages exposed to particulate TiO(2). The results of magnetometry, LDH release, and electron microscopy suggest that cytotoxicity of TiO(2) depends on the shape of the material.

Negative effect of photocopier toner on alveolar macrophages determined by in vitro magnetometric evaluation.

Photocopier toner has been implicated in the etiology of some pulmonary diseases. We examined here the in vitro toxicity of toner particles to alveolar macrophages. Cell magnetometry revealed that relaxation was not delayed in macrophages exposed to toner, which represents a rapid decrease in the remaining magnetism emitted by phagocytosed magnetite. However, relaxation was delayed in macrophages exposed to silica (positive controls). The release of intracellular LDH enzyme activity to the extracellular space was negligible in cells exposed to toner compared with negative and positive controls. Morphological examinations by light and electron microscopy revealed no abnormal findings in the exposed cells. A histochemical study using TUNEL staining and the electrophoretic profile of DNA obtained from cells exposed to toner and to silica were negative for apoptosis. The results of the present and other investigations into animal exposure indicate that photocopier toner is toxicologically inert. However, although the present study examined only effects in vitro, exposure to toner should be minimized because lung overloading in animals has been reported.

Cytotoxicity study of high temperature wool (HT wool) by cell magnetometric evaluation.

OBJECTIVES: We performed a cytotoxicity study by cell magnetometry, measured lactate dehydrogenase (LDH) activity by enzyme assay, detected DNA ladder formation, and performed morphological examination by electron microscopy in order to evaluate the safety of high temperature wool (HT wool), an asbestos substitute, using long and short chrysotile fibers (CF) as positive controls and phosphate buffered saline (PBS) as a negative control. METHODS: Alveolar macrophages were isolated from male Fisher rats. Following the addition of iron oxide particles (Fe(3)O(4)) to macrophages, HT wool, long or short CF was added. Then, the remanence strength was measured for 20 min after magnetization by an external field. Percent LDH release was calculated after determining LDH activity. DNA was detected using an apoptosis detection kit. Morphological observation was performed by taking electron micrographs of macrophages in the groups treated with HT wool and long- and short-CF. RESULTS: Rapid relaxation, an indicator of decay of cytotoxicity, was observed by cell magnetometry immediately after magnetization was ended in the groups treated with HT wool and PBS, showing that HT wool causes no harmful effect on the cytoskeleton. The CF-treated groups had higher LDH activity than the PBS- and HT wool-treated groups. No fragmentation of DNA was observed in any group. In morphological observation, cytotoxicity in macrophages was lower in the HT wool-treated groups than in the CF-treated groups. CONCLUSIONS: The results suggest that HT wool has no cytotoxicity, as evaluated by cell magnetometry, enzyme assay, DNA ladder detection and morphological examination.

Evaluation of hazardousness of refractory fibers by cell magnetometry.

This study was carried out to assess the cytotoxicity of three kinds of refractory fibers (RFs), RF1, RF2, and RF3, by cell magnetometry, lactate dehydrogenase (LDH) assay and morphological observation by scanning electron microscopy, using a mouse-derived cultured cell line, RAW264.7. As an indicator for cell magnetometry, Fe(3)O(4) was added to RAW264.7 cells. RF1, RF2 and RF3 were each added to an aliquot of this solution to make final concentrations of 250, 500 and 1,000 µg/ml in the experimental group. Phosphate buffered solution was added to make the control solution (n = 6). After culturing for 48 hr, the solution was magnetized from outside using a cell magnetometric apparatus, and the remnant magnetic field was measured for 20 min postmagnetization. In cell magnetometry, a significant delay of relaxation compared to that of the control was observed. In the LDH assay, LDH release into the culture medium was observed by addition of RFs. Furthermore, a quantity-dependent relationship was found between the quantity of RF added and the cytotoxicity in cell magnetometry and LDH assay. Morphological examination revealed incomplete phagocytosis of fibers and a decrease of microvilli in the experimental groups. These results suggest that RFs are cytotoxic to RAW264.7 cells, showing concentration-dependent cytotoxicity, and have a possible risk of cytotoxicity similar to that of asbestos. Further studies by pulmonary magnetometry are necessary to assess the hazardousness of RFs.

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

BACKGROUND: Asbestos 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. METHODS: Using 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 mum) 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. RESULTS: In 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 mum, 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. CONCLUSION: These 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.

Effects of language experience: neural commitment to language-specific auditory patterns.

Linguistic experience alters an individual's perception of speech. We here provide evidence of the effects of language experience at the neural level from two magnetoencephalography (MEG) studies that compare adult American and Japanese listeners' phonetic processing. The experimental stimuli were American English /ra/ and /la/ syllables, phonemic in English but not in Japanese. In Experiment 1, the control stimuli were /ba/ and /wa/ syllables, phonemic in both languages; in Experiment 2, they were non-speech replicas of /ra/ and /la/. The behavioral and neuromagnetic results showed that Japanese listeners were less sensitive to the phonemic /r-l/ difference than American listeners. Furthermore, processing non-native speech sounds recruited significantly greater brain resources in both hemispheres and required a significantly longer period of brain activation in two regions, the superior temporal area and the inferior parietal area. The control stimuli showed no significant differences except that the duration effect in the superior temporal cortex also applied to the non-speech replicas. We argue that early exposure to a particular language produces a "neural commitment" to the acoustic properties of that language and that this neural commitment interferes with foreign language processing, making it less efficient.

The cytotoxicity of microglass fibers on alveolar macrophages of fischer 344 rats evaluated by cell magnetometry, cytochemisry and morphology.

OBJECTIVES: The 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. METHODS: Alveolar 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. RESULTS: In 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. CONCLUSIONS: The cytotoxicity of MG on alveolar macrophages was demonstrated with cell magnetometry. The mechanism of the toxic effects of MG was related to cytoskeleton damage.

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.