Paleogenomics reveals independent and hybrid origins of two morphologically distinct wolf lineages endemic to Japan.

Little is known about the spatiotemporal dynamics of gray wolves in the Pleistocene across low-latitude regions of Eurasia. In Japan, a small-bodied endemic subspecies of Japanese wolves existed and went extinct in the early 1900s. The fossil record indicates that a giant wolf, which reached 70 cm in body height, inhabited Japan during the Pleistocene, but its evolutionary relationship, if any, with the Japanese wolf remains uncertain. Here, to reveal the genetic origin of the Japanese wolf, we analyzed ancient DNA from remains (recovered in Japan) of one Pleistocene wolf that lived 35,000 years ago and one Holocene wolf from 5,000 years ago. The analysis of the mitochondrial DNA revealed that the Pleistocene wolf was not part of the Japanese wolf clade but rather an earlier-diverging lineage. The analysis of the nuclear DNA of the Holocene Japanese wolf revealed that it was an admixture of the Japanese Pleistocene wolf and continental wolf lineages. These findings suggest that the Japanese wolf originated via waves of colonization of multiple Pleistocene wolf populations at 57-35 and 37-14 ka, respectively, followed by interpopulation hybridization.

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.

The increases in mRNA expressions of inflammatory cytokines by adding cleaning solvent or tetrachloroethylene in the murine macrophage cell line J774.1 evaluated by real-time PCR.

The use of a petroleum-derived cleaning solvent for dry cleaning, instead of tetrachloroethylene (perchloroethylene, PCE), has increased. The cleaning solvent may induce immunological alteration. In this study, murine macrophage-lineage J774.1 cells were exposed to the cleaning solvent at 0, 25, 50, and 75 µg/ml or PCE at 0, 400, 600, 800, and 1,000 µg/ml by vigorous vortexing. Cell viability was determined. The mRNA expressions of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), IL-6, IL-10, IL-12p40 (a dimer of IL-12), and IL-27p28 (a dimer of IL-27) were evaluated by real-time PCR. The mean viabilities in the 50 and 75 µg/ml groups of the cleaning solvent were significantly lower than that of the control. The mean mRNA expressions of TNF-α and IL-1ß in the 50 µg/ml group were significantly higher than those in the control. For PCE, the mean viabilities at 600 µg/ml and over were significantly lower than that of the control. The mean expressions of IL-6 and IL-10 in the 800 µg/ml group were significantly higher than that in the control. The productions of IL-1ß and TNF-α may be altered in human during intoxication of the cleaning solvent as well as those of IL-6 and IL-10 in human during that of PCE, and these may affect on immune cells.

Cytotoxicity and solubility evaluation of two types of whiskers by cell magnetometry.

OBJECTIVES: We investigated two types of whiskers, an antimony-containing tin-oxide-coated aluminum borate whisker (CABW) and an aluminum borate whisker (ABW), which are asbestos substitutes, in order to evaluate the safety of these fibers. METHODS: The cytotoxicity and solubility of CABW and ABW were evaluated by cell magnetometry, LDH assay and solubility test. RESULTS: ABW was found to be cytotoxic by cell magnetometry and slightly less soluble than CABW. In addition, it was found that the solubility of both fibers was intermediate between that of chrysotile and rock wool, as compared to our previous test results. Regarding the LDH assay, no significant difference was found among the fibers tested. These findings suggested that CABW, the surface of which is coated with antimony-containing tin oxide, had lower cytotoxicity and slightly higher solubility than ABW. CONCLUSIONS: This study was only a short-term cytotoxicity and solubility study. Therefore, further safety assessment should be carried out in long-term experiments to examine the half-life of these fibers and monitor the potential development of lung carcinoma or mesothelioma after intratracheal instillation of these fibers in rats.

Safety evaluation of rock wool after nasal inhalation in rats.

Asbestos is reported to cause pulmonary fibrosis, and its use has been banned. We examined the biopersistence and histopathological effect of rock wool (RW) fibers in rat lungs by a nose-only inhalation exposure study. Twenty-four rats were exposed to RW fibers for 6 h daily for 5 consecutive days. Six rats each were sacrificed shortly and 1, 2, and 4 wk after exposure, and the fiber numbers and sizes in lungs were determined. The fiber number in the lungs 4 wk after exposure significantly decreased. The clearance half time was 28 d for fibers with L>20 µm and 50 d for World Health Organization fibers. The reasons for the decrease in number and size of fibers include: 1) discharge outside of the body by mucociliary movement, 2) dissolution by body fluid, and 3) phagocytosis and digestion by alveolar macrophages. Twelve rats were used for histopathological examination, and the pathological changes were classified by Wagner's criteria. As a result, changes up to only Grade 2 were observed. The reason for the increase in macrophage number was considered to be a transient reaction of body defense. These results suggest that RW has low biopersistence and only a limited pathological effect.

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.

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.

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.

Behavior of rock wool in lungs after exposure by nasal inhalation in rats.

To evaluate the safety of rock wool (RW fibers), we examined the biopersistence of a RW sample in the lungs of rats, based on the changes of fiber number and fiber size in terms of length and width, by a nose-only inhalation exposure study. Twenty male Fischer 344 rats (6-10 weeks old) were exposed to RW fibers at a concentration of 70 (21) fiber/m(3) and 30 (6.6) mg/m(3), arithmetic mean (geometric standard deviation), continuously for 3 h daily for five consecutive days. Five rats each were sacrificed shortly and at 1, 2, and 4 weeks after exposure, and their lung tissues were ashed by a low-temperature plasma-asher. Then, the numbers and sizes of fibers in the ashed samples were determined using phase-contrast microscope and computed image analyzer. The fiber numbers in the lungs 4 weeks after exposure significantly decreased from the baseline value, i.e., shortly after exposure (P < 0.05). The half-lives of RW fibers calculated from the one-compartment model were 32 days for total fibers and 10 days for fibers longer than 20 mum. The decrease of fiber number was 53.6% by 4 weeks after exposure (baseline group = 100%). Likewise, fiber sizes significantly decreased by 4 weeks after exposure (P < 0.05), probably because fibers were dissolved in body fluid, ingested by alveolar macrophages or discharged to outside of the body by mucociliary movement. In future studies, it is necessary to examine the long-term persistence of RW fibers in the lungs.

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.

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.

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/m(3) 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/cm(3), and 29.9 (28.3) mg/m(3), 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 microm, 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).

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.

Behavior of rock wool in rat lungs after exposure by nasal inhalation.

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/m(3) and a dispersion density of 30.4 (6.6) mg/m(3) [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 microm 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.

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

OBJECTIVES: Previous 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 Al(2)O(3) and a lower concentration of SiO(2), 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. METHODS: Male Fischer 344 rats were exposed to fibers at the target exposure concentration of 30 mg/m(3) 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. RESULTS: The 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. CONCLUSIONS: Both 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.

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.

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.