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

Environ Health Prev Med (2011) 16:327–334 DOI 10.1007/s12199-010-0203-9 REGULAR ARTICLE Cytotoxicity and solubility evaluation of two types of whiskers by cell magnetometry Yuichiro Kudo • Yoshiharu Aizawa Received: 27 August 2010 / Accepted: 3 December 2010 / Published online: 21 January 2011 Ó The Japanese Society for Hygiene 2011 Abstract 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. Keywords Cell magnetometry  Relaxation  Whisker  Cytotoxicity  Solubility Y. Kudo (&)  Y. Aizawa Department of Preventive Medicine and Public Health, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami, Sagamihara, Kanagawa 252-0374, Japan e-mail: Introduction Asbestos has been widely used as an industrial material because of its high fire and heat resistance and chemical stability. Many in vitro and in vivo studies on the hazards of asbestos have been reported [1, 2]. A relationship between asbestos and the development of lung carcinoma and mesothelioma is well documented [3, 4]. In Japan, the use and manufacturing of white asbestos was prohibited in principle in 2004, and a policy to totally ban the use of asbestos by 2008 was established. Taking this trend into consideration, the development and use of asbestos substitutes in industry have been hastened. Currently, many types of man-made mineral fibers have been developed and are widely used as asbestos substitutes, such as building materials and friction materials in brakes. Not only asbestos, but also fibers with a width of less than 0.25 lm and a length greater than 8 lm have been reported to exert especially high carcinogenicity, and fibers of small width, long length and low solubility within the human body may be carcinogenic [5–7]. Whisker, which is a man-made crystalline fiber, is a type of asbestos substitute and is defined as a single crystal with a cross-sectional area of 8 9 10-5 cm2 or less and a length of 10 times the average cross-sectional diameter or more. Whiskers include aluminum borate whisker, potassium octatitanate whisker (PT) or silicon carbide whisker (SiC). The two types of whiskers, antimony-containing tin-oxidecoated aluminum borate whisker (CABW) and aluminum borate whisker (ABW), used in our study have high intensity, hardness, heat resistance, electrical insulation and chemical resistance. Hence, they are widely used as materials for resin/metal reinforcement, friction, insulation, refractory and heat insulation, etc. Because of such properties, they are expected to be used more extensively in the 123 328 future. However, they have been used industrially only recently, with a lower number of safety reports than for other asbestos substitutes. There is therefore an urgent need to evaluate the safety of these whiskers. In this study, the cytotoxicity of CABW and ABW was evaluated by cell magnetometry and lactate dehydrogenase (LDH) enzyme assay using alveolar macrophages, which play a primary role in the defense mechanism against the invasion of foreign matter in the respiratory tract. Further, solubility was examined using physiological saline or simulated body fluid (Gamble’s solution), and compared with that of chrysotile fibers and rock wool (RW) fibers, whose solubility was previously examined in our laboratory [8]. Materials and methods Test substances CABW and ABW, both provided by Konica Minolta Business Expert Inc. (Tokyo, Japan), were used as test substances. The mean length of both substances was 10–30 lm and mean width 0.5–1.0 lm. CABW was chemically composed of 60–80% aluminum borate, 16–38% tin oxide, 2–5% titanium dioxide and 2–4% antimony trioxide, and ABW was composed of 97% aluminum borate. After sterilization by an autoclave (1.1 atmospheric pressure, 121°C, 20 min), these test substances were used after suspension in phosphate-buffered saline (PBS) at pH 7.4. Alveolar macrophage preparation Six male Fisher rats (F344/N Slc) weighing 200–250 g were used. Animals were anesthetized by intraperitoneal injection of sodium pentobarbital (100 mg/kg) and killed by exsanguination from the abdominal aorta after making a midline abdominal incision. With the trachea exposed, a silicon tube (Atom Intravenous Catheter, 5 French for cutdown, Atom Medical Corp., Tokyo, Japan) was inserted and fixed. Bronchoalveolar lavage (BAL) was carried out by infusing 4 ml PBS, pH 7.4, containing sterilized 0.1% ethylenediamine tetraacetic acid followed by recovery of the BAL fluid (BALF). This procedure was performed approximately 10 times in total. The BALF obtained was pooled and subjected to centrifugation at 1,800 rpm for 10 min, and the pellet was suspended in serum-free culture medium (Macrophage-SFM liquid, Life Technologies Inc., Rockville, MD). A portion of the suspension was used for determination of cell counts with a hemocytometer after Trypan Blue staining. Cells with a survival rate of 90% or more were used in this study. 123 Environ Health Prev Med (2011) 16:327–334 The alveolar macrophage suspension prepared by this procedure was diluted with Macrophage-SFM liquid, yielding an aliquot of 1 9 106 cells per well. Each aliquot was poured into a well, with a cell disc of 1 cm diameter. Cell magnetometry Cell magnetometry was performed in the same manner as reported by Keira et al. [9]. As an indicator for cell magnetometry, Fe3O4 particles suspended in PBS were added at a final concentration of 50 lg/ml in each well prepared according to the procedure described above. Then, the two test substances were added in the experimental groups at a final concentration of 40, 80 or 160 lg/ml, and 50 ll PBS was added to the negative control group. These test samples were cultured in a 5% CO2 incubator at 37°C for 18 h. After culturing, the glass disc harboring adherent alveolar macrophages was extracted from each well and placed in a glas (...truncated)