Fluoride Vol. 32 No. 3 153-161 1999 Research Report 153 EFFECTS OF FLUORIDE AEROSOL INHALATION ON MICE Xue-Qing Chen,a,b Kazuhiko Machidab and Mitsuru Andoa Tsukuba, Japan SUMMARY: The effects of fluoride aerosol inhalation on mice were studied using an inhalation chamber. Five-week-old male ICR mice were exposed to airborne fluoride (13.3 mgF/m 3) 4 hr per day for 10, 20 or 30 days. Significant differences in relative lung weight were observed between the exposed groups and the control. No significant changes were found in relative kidney weight and body weight of the exposed mice. Bone fluoride retention and urinary fluoride excretion increased with exposure time. Keywords: Airborne fluoride, Fluoride aerosol, Fluoride-exposed mice, Fluoride inhalation. INTRODUCTION Overexposure to fluoride causes toxicity in animals and humans. Excessive fluoride exposure during the period of tooth development may result in defective tooth formation, and intake of elevated levels of fluoride over prolonged periods of time may result in skeletal fluorosis. Drinking water containing high concentrations of fluoride is the major cause of dental fluorosis and skeletal fluorosis. In some areas of the world, e.g., China and India, there are a large number of people with dental fluorosis and skeletal fluorosis caused by drinking water containing high levels of fluoride. 1-4 Fluoride is also found in a wide range of concentrations in coal, a main fuel energy source for industrial and domestic processes in China. In some districts, especially in rural areas, coal containing high concentrations of fluoride is used for cooking and crop drying. Indoor burning of coal for cooking and crop drying with poor ventilation results in indoor air pollution. 5-7 In 1997, it was reported that there were 16.5 million people with dental fluorosis and 1.08 million people with skeletal fluorosis in endemic areas of coal burning in China. 8 Although effects of occupational fluoride exposure on humans have been reported, there is limited information about the effects of fluoride inhalation on animals and humans because of the difficulties in carrying out exposure experiments. 9-11 In this study, we exposed mice to airborne fluoride in an inhalation exposure chamber. The potential effects of fluoride inhalation on the animals, including food consumption, body weight, organ weight, urinary fluoride excretion, and fluoride retention in bone, were investigated. MATERIALS AND METHODS Animals and Diet: Five-week old, SPR-grade male ICR mice (Clea, Japan Inc., Tokyo) were housed in plastic cages kept at 22±1°C, relative humidity of 50±10%, and a light-dark cycle of 12 hr. The animals were divided into 6 groups of 6 each (3 exposure and 3 control groups). Mice were fed an AIN93M purified diet containing low fluoride (1.0 F mg/kg, diet) and tap water ad libitum. The diet (Oriental Yeast Co. Ltd. Tokyo, Japan) contained recom——————————————— aFor Correspondence: X-Q Chen, Regional Environment Division, National Institute for Environmental Studies, 16-2 Onogawa Tsukuba, Ibaraki 305-0053 Japan. Tel/fax: +81298-502395 E-mail: chen.xueqing@nies.go.jp; bGraduate School of Human Sciences, Waseda University, Tokorozawa, Saitama, Japan. 154 Chen, Machida, Ando mended amounts of vitamins, minerals and other nutrients in α-cornstarch, 155 g/kg; cornstarch, 466 g/kg; milk casein, 140 g/kg; sugar, 100 g/kg; soybean oil, 40 g/kg; and cellulose, 50 g/kg. Exposure System: A 0.256-m3 inhalation exposure chamber was used in the study. The chamber and atomisation system were constructed of stainless steel, tetrafluoroethylene and glass. Fluoride aerosol was generated using an atomiser system (Sibata Co., Tokyo, Japan) by atomising 0.1 M NaF solution followed by dehydrating the aerosol to form submicron particles. The fluoride aerosol concentrations were monitored continuously by a Digital Dust Indicator (Model PCD-1, Sibata Co., Tokyo, Japan) during exposure. The animals were transferred to stainless-steel cages and exposed to the sodium fluoride (NaF) aerosol in the chamber 4 hr per day for 10, 20 or 30 days. After the fluoride exposure, distilled water was provided for 30 min to clean the chamber and animals. The positions of cages within the inhalation chamber were rotated daily. Food and water were not provided to the mice during the 4-hr exposure. Sampling and Fluoride Analysis: Urine samples of the mice were collected by using metabolism cages. A 0.5-1.0-mL a urine sample from each mouse was mixed with 1.0 mL of total ionic strength adjustment buffer (TISAB III, Orion Research Inc., Boston, USA), and then diluted to 10.0 mL with distilled deionized water. The fluoride aerosol concentrations in the chamber were monitored by a PCD-1 digital dust indicator during exposure and sampled by use of a newly developed Andersen type AND sampler (Sibata Co., Tokyo, Japan) with 19 and 35 mm T60A20 filters (Pallflex Prod. Corp., Putnam, Conn., USA) for 10-30 min at a flow rate of 2.5 L/min. 12 Before sampling, the filters were washed twice with distilled deionized water for 10 min with an ultrasonic cleaner to decrease background fluoride levels, and then dried at 50°C. The sampled filter was cut into pieces and placed in a mixture made up of 1.0 mL of TISAB III solution and 9.0 mL of deionized distilled water. Fluoride content in the diet was determined by a micro diffusion method. In this procedure, 0.1-1.0 g of powdered sample was accurately weighed in a plastic petri dish (Falcon, Becton Dickinson, New Jersey, USA). Then, 2.0 mL of distilled deionized water was added. Vaseline was applied to the inside rim of the petri dish cover, and 50 µL of 0.05 N NaOH solution was placed in five separate drops on the inside cover of petri dish. 1.0 mL of 1.5 M H 2SO4HMDS (hexamethyldisiloxane, C6H18OSi2) saturated solution was then added through a hole of the cover. The hole was immediately sealed with Vaseline. After diffusion overnight at room temperature, 25 µL of 0.15 M acetic acid was added to the NaOH drops on the petri dish lid. The NaOH drops were collected with pipette, and deionized distilled water was added to give a final volume of 100 µL. The fluoride concentration in each sample was determined with a fluoride electrode (see below). After exposure for 10, 20 and 30 days, the animals were weighed, anesthetized by intraperitoneal injection of sodium pentobarbital and sacrificed by severing the main abdominal artery. The lung, liver and kidney were removed, Fluoride 32 (3) 1999 Effects of fluoride aerosol inhalation on mice 155 washed with 0.9% NaCl solution, weighed immediately, and stored at -40°C. The leg bones were collected, and the muscles were removed and incubated at 37°C in Papain solution (1:350 w/v) for 24 hr. The bones were washed with distilled water, dried at 105°C for 2 hr, heated at 300°C for 3 hr, and then at 600°C for 6 hr in an electric furnace (Model TMF-1100, Tokyo-Rika Co., Tokyo, Japan). The fat-free bones were ground into powder, and 0.02 g of the powder was accurately weighed into a test tube, followed by the addition of 5.0 mL of 0.25 M HCl solution. The mixture was stored overnight at room temperature. About 0.2 mL of 0.05% bromophenol blue solution was added, the pH of the solution was adjusted to 5.2-5.6 with 0.25 M NaOH solution. Finally, 2.0 mL TISAB III solution was added and the sample was diluted to 20.0 mL with deionized distilled water. Fluoride concentrations in all samples were determined with a model 9609BN combination fluoride ion selective electrode and a 720A pH/ISE Meter (Orion Research, Boston, USA). Freeze-dried urine 2671a (U.S. Department of Commerce, National Institute of Standards and Technology, MD, USA) was used as standard reference material for fluoride analysis. The results are shown in Table 1. TABLE 1. Fluoride concentration in standard reference material (mg/L) SRM-2671a Determined values Low level Elevated level 0.56 ± 0.02 (0.53 - 0.58) 5.5 ± 0.1 (5.3 - 5.6) Certified values 0.55 ± 0.03 5.7 ± 0.3 Values are means ± SD, n=9. Powdered bone samples were digested with 1 mL of a mixture of 60% HNO3 and 61% HClO4 (3:1, v/v) in a Pyrex test tube and heated at 50°C for 2 hr, and then at 105°C overnight with a Teflon-ball. The sample was then heated at 140°C for 6 hr without the Teflon-ball, and at 180°C for 2 hr. The digest was diluted to 5.0 mL with deionized distilled water and the Ca, Zn, Mg, Fe and Cu concentrations of the digested samples were determined by inductively coupled plasma atomic emission spectrometer (ICP-AES) (Model Spectro Flame-EOP, Spectro Analytical Instruments, Germany). Statistical Analysis: The results were analyzed for significant differences between groups using Student’s t test. Differences with p <0.05 were considered significant. RESULTS Fluoride concentrations in distilled water, CE-2 commercial diet, and AIN93M purified diet (Clea Co., Tokyo, Japan) were 0.007±0.001 mg/L, 57.3±6.7 mg/kg, and 1.0 mg/kg, respectively. Fluoride 32 (3) 1999 156 156 Fluoride concentrations in the exposure chamber (mg/m 3 ) Chen, Machida, Ando Fluoride 32 (3) 1999 10.00 9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 < 2 µm 1.00 2-10 µm >10 µm 0.00 1 2 3 4 5 6 7 Number of sampling Figure 1. Distribution of aerodynamic diameter in the chamber 8 Effects of fluoride aerosol inhalation on mice 157 The mean total fluoride aerosol concentration in the chamber was 13.30±1.30 mg/m3. The fluoride aerosol concentrations in the chamber were stable during the exposure period, and the distribution of aerodynamic diameter (AD) in the chamber showed that greater than 99.7% of the fluoride aerosol contained particles that were respirable ≤10 AD), and that more than 56.3% of the particles had diameters of less than 2 m (Figure 1). The diet consumption of the mice was measured continuously during the experiment. The daily feed intake of animals exposed to NaF was 4.0±0.6 g/mouse/day, while the value for the control was 3.6±0.7 g/mouse/day. T-tests indicated that the mean daily diet consumption between the control and the exposed animals was not significantly different from each other. Animals exposed to fluoride for 30 days were weighed twice per week during the experiment. As shown in Figure 2, the fluoride inhalation had no significant effect on body weight. Significant differences in relative lung weight were observed in all exposed groups as compared to the control group. A significant decrease in relative liver weight was observed only in the 30-day exposed group. No significant changes were found in relative kidney weight of all exposed groups (Table 2). TABLE 2. Effect of fluoride inhalation on relative organ weights Organ Weight / Body Weight (%) Lung Liver Kidney Time Group 10 days Control F-exposed 0.58 ± 0.05 0.64 ± 0.05* 6.11 ± 0.70 6.00 ± 0.37 1.69 ± 0.19 1.71 ± 0.11 20 days Control F-exposed 0.57 ± 0.02 0.65 ± 0.03† 5.21 ± 0.28 5.35 ± 0.79 1.52 ± 0.15 1.64 ± 0.22 30 days Control F-exposed 0.54 ± 0.03 0.58 ± 0.02† 5.98 ± 0.42 5.40 ± 0.36* 1.55 ± 0.09 1.52 ± 0.17 Values are means ± SD. n=5-6. *p<0.05 and †p<0.01. Urinary fluoride excretion decreased from 17.8 to 6.87 µg F per day after 5 days of feeding purified diet and decreased continually to 5.36 µg F after 10 days of feeding purified diet. Inhalation exposure was started on the 25th day after feeding purified diet. In the exposed animals, urinary fluoride excretion significantly increased with exposure time, and was 4.34, 19.6 and 29.2 µg F per day after 3, 15 and 23 days of inhalation exposure, respectively (Figure 3). A linear correlation was found between inhaled fluoride levels and urinary fluoride excretion (r =0.936, p<0.01, n=10). Significant increases in fluoride concentrations in the bone of the exposed mice were also observed. The fluoride concentration in leg bone exceeded 775 mg/kg in mice exposed to NaF for 10 days, which was 60% higher than that of the control. In mice exposed to NaF for 20 or 30 days, the bone fluoride levels were 955 and 1276 mg/kg, respectively, which were approximately two and three times higher than those of the control animals (Figure 4). Fluoride 32 (3) 1999 158 Chen, Machida, Ando The concentrations of Cu, Zn and Fe in bone significantly decreased, and no significant differences in Ca and Mg concentrations in bone were observed in the exposed mice (Table 3). TABLE 3. Concentrations of trace elements in bone Ca (mg/g) Cu (mg/kg) Zn (mg/kg) Fe (mg/kg) Mg (mg/kg) Time Group 10 days Control F-exposed 459 ± 56 420 ± 23 4.02 ± 0.62 248 ± 15 2.18 ± 0.23‡ 168 ± 5‡ 107 ± 14 7759 ± 1150 81 ± 7† 6689 ± 330 30 days Control F-exposed 354 ± 25 376 ± 22 4.00 ± 0.20 228 ± 18 3.26 ± 0.60* 212 ± 14 150 ± 21 5868 ± 632 127 ± 7* 5704 ± 222 Values are means ± SD, n=5-6. *p<0.05, †p<0.01, and ‡p<0.001 compared with the control. Control F-exposed Body weight (g/mouse) 45 40 35 30 0 1 3 6 9 12 15 18 21 25 Exposure time (days) Figure 2. Growth curves of control and exposed mice. Values are means ± SD. n= 6. Fluoride 32 (3) 1999 30 31 159 Urinary fluoride excretion (µg/day) Effects of fluoride aerosol inhalation on mice Time of low fluoride diet fed (days) Figure 3. Urinary excretion of fluoride during the experiment. Exposure was started on the 25th day of low-F diet fed. Fluoride concentrations in bone (mg/kg) 1500 Control *** F-exposed 1200 *** 900 *** 600 300 0 10 20 Exposure time (days) 30 Figure 4. Bone fluoride accumulation in control and exposed mice. Values are mean ± SD. n= 5 - 6. ***p< 0.001. Fluoride 32 (3) 1999 160 Chen, Machida, Ando DISCUSSION Normally, commercial diet contains high fluoride levels because of the presence of powdered fish or bone containing high fluoride. To study the effect of fluoride inhalation on animals, mice were fed a low-fluoride diet for 20 days before starting the exposure in order to reduce the fluoride burden in the body. As shown in Figure 3, the urinary excretion of fluoride rapidly decreased after purified diet was fed and significantly increased with exposure time. A linear correlation was found between exposure time and urinary excretion of fluoride. However, the urinary excretion of fluoride decreased from 29.2 mg F on the 23rd exposure day to 21.8 mg F per day when the exposure was ended. Several factors may influence the urinary excretion of fluoride, such as total current intake, previous exposure to fluoride, age, urinary flow, urine pH, and kidney status. The decrease in urinary fluoride excretion may be due to kidney damage caused by fluoride inhalation. Although slight increases in feed intake were observed in exposed animals, no statistically significant changes were observed as indicated by Student’s t test. It has been reported that there is no effect of fluoride exposure on body weight of animals by Yoshida et al,13 and by Boros et al.14 However, when exposure to high levels of fluoride has occurred, reductions in body weight gain were observed in lambs by Vashishtha et al.15 Slight body weight decrements were noted in groups of vinyl fluoride-exposed rats and mice.16 Body weight reductions were also observed in rats after exposure to 1300 ppm HF (~ 823 mg/m3) for 30 min.17 Characterizing particle size provides estimates of the deposition efficiency and pattern in the test animals because deposition in the respiratory tract is dependent on particle size. The smaller the particle diameter is, the higher is the deposition fraction in the lung. 11 In our study, 99.7% of fluoride aerosol in the exposure chamber contained particles of respirable mean diameters less than 10 m, and more than 55% of fluoride aerosol was less than 2 m AD particles. The relative lung weights increased significantly in exposed animals, and the results were similar to those of our previous histochemical study (reported at the XXth conference of the International Society for Fluoride Research in 1994), suggesting that sodium fluoride inhalation causes lung edema in exposed animals.18 This work was presented at the XXIInd Conference of the International Society for Fluoride Research, Bellingham, Washington, USA, Aug. 24-27, 1998. ACKNOWLEDGMENTS The authors are grateful to Dr. Warren T. Piver of the National Institute of Environmental Health Sciences (USA) for his critical reading of the manuscript, and to Mr. K. Tamura and Ms. M. Tadano of the National Institute for Environmental Studies for their technical assistance in the study. Fluoride 32 (3) 1999 Effects of fluoride aerosol inhalation on mice 161 REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Wang J-P, Yang C-Z and Xu X-F. 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