Fluoride Vol. 32 No. 3 153-161 1999 Research Report
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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.
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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.
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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).
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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.
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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.
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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.
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Effects of fluoride aerosol inhalation on mice
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Published by the International Society for Fluoride Research
Editorial Office: 17 Pioneer Crescent, Dunedin 9001, New Zealand
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Fluoride 32 (3) 1999