55, 116 –123 (2000)
Copyright © 2000 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
1-Bromopropane, an Alternative to Ozone Layer Depleting Solvents,
Is Dose-Dependently Neurotoxic to Rats
in Long-Term Inhalation Exposure
Gaku Ichihara,* ,1 Junzoh Kitoh,† Xiaozhong Yu,‡ Nobuyuki Asaeda,§ Hisakazu Iwai,§ Toshihiko Kumazawa,§ Eiji Shibata, ¶
Tetsuya Yamada,* Hailan Wang,* Zhenlin Xie,* and Yasuhiro Takeuchi*
*Department of Occupational and Environmental Health, Graduate School of Medicine, and †Institute for Laboratory Animal Experiments, School of
Medicine, Nagoya University, Nagoya, Japan; ‡National Institute of Industrial Health, Kanagawa, Japan; §Safety Assessment Laboratory, Sanwa Kagaku
Kenkyusho Co. Ltd., Mie, Japan; and ¶Department of Medical Technology, School of Health Sciences, Nagoya University, Nagoya, Japan
Received September 13, 1999; accepted December 22, 1999
1-Bromopropane has been newly introduced as an alternative
to ozone layer-depleting solvents. We aimed to clarify the
dose-dependent effects of 1-bromopropane on the nervous system. Forty-four Wistar male rats were randomly divided into 4
groups of 11 each. The groups were exposed to 200, 400, or 800
ppm of 1-bromopropane or only fresh air 8 h per day for 12
weeks. Grip strength of forelimbs and hind limbs, maximum
motor nerve conduction velocity (MCV), and distal latency
(DL) of the tail nerve were measured in 9 rats of each group
every 4 weeks. The other 2 rats of each group were perfused at
the end of the experiment for morphological examinations. The
rats of the 800-ppm group showed poor kicking and were not
able to stand still on the slope. After a 12-week exposure,
forelimb grip strength decreased significantly at 800 ppm and
hind limb grip strength decreased significantly at both 400 and
800 ppm or after a 12-week exposure. MCV and DL of the tail
nerve deteriorated significantly at 800 ppm. Ovoid or bubblelike debris of myelin sheaths was prominent in the unraveled
muscular branch of the posterior tibial nerve in the 800-ppm
group. Swelling of preterminal axons in the gracile nucleus
increased in a dose-dependent manner. Plasma creatine phosphokinase (CPK) decreased dose-dependently with significant
changes at 400 and 800 ppm. 1-Bromopropane induced weakness in the muscle strength of rat limbs and deterioration of
MCV and DL in a dose-dependent manner, with morphological
changes in peripheral nerve and preterminal axon in the gracile
nucleus. 1-Bromopropane may be seriously neurotoxic to humans and should thus be used carefully in the workplace.
Key Words: 1-bromopropane; neurotoxicity; alternative to chlorofluorocarbons; neuropathy; motor nerve conduction velocity;
Parts of this study on the neurotoxicity of 1-bromopropane were presented
in the 71st and 72nd Annual Meetings of the Japan Society for Occupational
Health (Ichihara et al., 1998b, 1999b) and the 9th Annual Meeting for the study
of the peripheral nerve (Ichihara et al., 1998c).
1
To whom correspondence should be addressed at the Department of
Occupational and Environmental Health, 65 Tsurumai-cho, Showa-ku, Nagoya
University Graduate School of Medicine, Nagoya, Japan. Fax: (81) (52)
744-2126. E-mail: [email protected].
distal latency; tibial nerve; Wallerian-like degeneration; preterminal; gracile nucleus; creatine phosphokinase.
1-Bromopropane was recently introduced as an alternative to chlorofluorocarbons and 1,1,1-trichloroethane,
which have the potential to destroy the ozone layer. This
new alternative has less ozone depleting potential, yet has
the high volatility and nonflammability required for a cleaning agent. It has come to be a very important solvent,
especially after 2-bromopropane was phased out after its
reproductive and hematopoietic toxicity to humans were
revealed (Ichihara et al., 1999a; Kim et al., 1996; Park et al.,
1997) and rats (Ichihara et al., 1996, 1997; Kamijima et al.,
1997a,b; Lim et al., 1997; Nakajima et al., 1997a,b; Yu et
al., 1997, 1999b). Following these studies, we clarified the
neurotoxicity of 2-bromopropane in rats because polyneuropathy was also reportedly found in Korean workers (Yu et
al., 1999a). At the same time, we did a preliminary examination of the neurotoxicity of 1-bromopropane at 1000
ppm, and we unexpectedly found that, after 4 weeks of
exposure (Yu et al., 1998), 1-bromopropane induced severe
paralysis of hind limbs, decreased maximum motor nerve
conduction velocity (MCV), and increased distal latency
(DL). The same concentration of 2-bromopropane did not
induce paralysis and caused little change in MCV or DL (Yu
et al., 1999a). These studies suggested that 1-bromopropane
might have more potent neurotoxicity than 2-bromopropane.
However, our preliminary experiment (Yu et al., 1998) on
neurotoxicity of 1-bromopropane had a limitation, i.e., the
exposure was done only at a single concentration of 1000
ppm and was discontinued after 4 –7 weeks because of rat
debilitation.
The present experiment aimed to clarify the concentrationand exposure period-dependent neurotoxicity of 1-bromopropane and its morphological characteristics.
116
117
NEUROTOXICITY OF 1-BROMOPROPANE
MATERIALS AND METHODS
Animals and Exposure
A total of 44 Wistar male rats (specific pathogen-free, 9 weeks old) were
purchased from Shizuoka Laboratory Animal Center, Japan. They were housed
and acclimated to their new circumstances for 1 week. Eight rats were
randomly selected and divided into 4 groups of 2 each for morphological
studies. These rats were exempted from measurement of electrophysiological
indices of the tail nerve or grip strength, because physiological stimulation of
the tail or limbs might cause morphological artifacts of the axons in the gracile
nucleus. The remaining 36 rats were randomly divided into 4 groups of 9 each
with a stratified sampling method based on body weight and motor nerve
conduction velocity (MCV). These rats were used for evaluation of grip
strength, maximum motor nerve conduction velocity (MCV), distal latency
(DL), organ weights, and blood biochemical indices. The 4 groups of 11 each,
which consisted of 2 rats for morphological examination and 9 rats for
quantiative evaluation, were exposed to 200, 400, or 800 ppm 1-bromopropane
or fresh air, respectively in stainless steel chambers (884 ⫻ 884 ⫻ 1000 mm).
The highest concentration used was 800 ppm because our previous study
showed that the rats exposed to 1000 ppm 1-bromopropane were debilitated
after 4 weeks (Yu et al., 1998). 1-Bromopropane exposure lasted from 1400 h
to 2200 h, 8 h a day, for 12 weeks. The internal size of a cage was 260 ⫻ 380 ⫻
180 mm. Food and water was provided ad libitum. The environment was kept
on a 12-h light-dark cycle (lighting on at 0900 h and off at 2100 h) and was
held at 23–25°C and 57– 60% humidity. Body weight was measured between
10:00 and 11:00 A.M. once a week. The inhalation exposure system used in the
present study was described elsewhere (Ichihara et al., 1997; Takeuchi et al.,
1989). In brief, the regulated volume of solvents was evaporated at room
temperature and mixed with a larger volume of clean air to achieve the desired
concentration. The vapor concentrations were measured every 10 s by gas
chromatography and controlled to within ⫾ 5% of the target concentration by
means of a personal computer. Rectification boards, which had numerous small
holes, were positioned at the entrance to the chamber for the vapor to be
distributed uniformly. The concentrations at 9 points were within ⫾ 3% of the
value of the central point in the chambers. 1-Bromopropane (99.81% purity in
the chart area of a capillary gas chromatograph with flame ionization detector
[FID]) was kindly supplied by Tosoh Co., Japan. The structure of 1-bromopropane was confirmed with proton nuclear magnetic resonance (NMR). All of the
1-bromopropane used was from the same lot. Repeated gas chromatographic
analyses showed no change in the composition of 1-bromopropane in storage
bottle or its vapor in the chambers throughout the experiments. Japanese law
concerning the protection and control of experimental animals and the “Guide
of Animal Experimentation,” Nagoya University School of Medicine, were
followed throughout the experiments.
in diameter were used. Electrode A was located 3 cm distal from the anus,
electrode C was 3– 4 cm proximal from the end of the tail, and electrode B was
5 cm proximal from electrode C. The electrodes were inserted tangentially into
the subcutanea to prevent injury to the nerve trunks. The tail was immersed in
a paraffin bath kept at 37.0 to 37.5°C. The nerve conduction velocity was
measured no sooner than 4 min and no later than 20 min after immersion. The
tail nerve was stimulated at electrode A or B by a square pulse of 0.3-ms
duration and supramaximal strength, using an electrostimulator equipped with
an electroisolator (SEN-7103, Nihon Kohden). The biopotentials were observed at electrode C with an Addscope (ATAC-350, Nihon Kohden). MCV
(AB/latency time [AC-BC]) and DL (latency time [BC]) were measured before
the start of exposure and every 4 weeks during exposure. Conduction velocities
were measured more than 10 h after the end of the exposure to remove the
acute effects of inhalation.
Brain Weights and Blood Biochemical Indices
After 12 weeks of exposure to 1-bromopropane, 9 rats of each group were
killed under pentobarbital anesthesia by collecting all blood with heparinized
syringe through the abdominal aorta. The cerebrum, cerebellum, and brainstem
were dissected out and weighed. Plasma was separated by centrifugation at
room temperature, and stored at – 80°C until analysis. Plasma GOT (glutamicoxaloacetic transaminase), GPT (glutamic-pyrubic transaminase), LDH (lactate dehydrogenase), ALP (alkaline phosphatase), CPK (creatine phosphokinase), BUN (blood urea nitrogen), creatinine, uric acid, total bilirubin, glucose,
triglyceride, free fatty acid, total cholesterol, phospholipid, total protein, albumin, globulin, Ca (calcium), and Pi (inorganic phosphate) were measured with
a Hitachi model 736 –10 Auto Analyzer.
Morphological Examination of Peripheral Nerve, Central Nerves and
Muscles
After 11 weeks, the walking status of the rats on the horizontal plane and a
slope of 25° was observed and videotaped. The surfaces of the plane and slope
were covered with coarse-textured cloth so that the control rats could walk or
climb easily.
Two rats from each group were perfused from the left ventricle with two
different kinds of fixatives after the 12-week exposure, one with 10% buffered
formalin (pH 7.2) and the other with Zamboni’s solution. Muscular branches
of the posterior tibial nerve, the brain, and the lumbar enlargement of the spinal
cord were dissected out from the rat perfused with buffered formalin. Muscular
nerves were dissected out for unraveled specimens because we observed a
remarkable weakness of muscle. They were post-fixed in 0.5% osmium tetroxide for 15 min, immersed in 30% ethanol for 60 min, and then transferred
into 50% glycerin. Specimens were loosened and unraveled by needles in the
50% glycerin under a binocular dissection microscope. The brain was cut
transversely at the level of the optic chiasma, the caudal margin of the
mamillary body, the paraflocculus of the cerebellum (corresponding to caudal
pole of the pons), and the gracile nucleus of the medulla oblongata. The blocks
of the brain and the spinal cord were embedded in paraffin, and several serial
sections were made around the levels mentioned above. The sections were
stained by the Klüver-Barrera method for light-microscopic observation. Small
tissue blocks of tibial nerve, brain, and soleus muscle perfused with Zamboni’s
solution were embedded in epoxy resin and cut into semi-thin or ultra-thin
sections for light or electron microscopic observation. The tibial nerve and its
branches were dissected out with their neighboring muscle tissue to prevent
physical damage from the dissection process.
Forelimb and Hind Limb Grip Strength
Statistical Analysis
Forelimb and hind limb grip strength was measured from 9:00 A.M. to 11:00
before the start of exposure and every 4 weeks. The grip strength was
measured using a push-pull scale (Imada Co., Ltd., Japan) according to
Meyer’s method (Meyer et al., 1979).
Multiple comparisons were made between the exposure groups and the
control in the means of body weights, organ weights, grip strength, MCVs, and
DLs using Dunnett’s method following one-way analysis of variance
(ANOVA). A probability ( p) of ⬍ 0.05 was accepted as statistically significant.
Walking Status of Rats
A.M.
Electrophysiological Examination of Tail Nerve of Rats
MCV and DL were measured in the rats’ tails every 4 weeks, in accord with
our previous studies (Ichihara et al., 1998a; Ono et al., 1979; Takeuchi et al.,
1980; Yu et al., 1999a; Yu et al., 1998). The rats were wrapped in a towel to
keep them immobilized without anesthesia. Stainless steel electrodes 0.34 mm
RESULTS
The concentrations of 1-bromopropane in the 3 chambers
were 208 ⫾ 15, 412 ⫾ 24, and 821 ⫾ 38 (mean ⫾ SD) ppm,
118
ICHIHARA ET AL.
TABLE 1
Time Course of Body Weight, Forelimb Grip Strength, and Hind Limb Grip Strength
Week
Body weight (g)
Control (8)
200 ppm (9)
400 ppm (9)
800 ppm (9)
Forelimb grip strength (mg)
Control (8)
200 ppm (9)
400 ppm (9)
800 ppm (9)
Hind limb grip strength (mg)
Control (8)
200 ppm (9)
400 ppm (9)
800 ppm (9)
0
4
8
12
307 ⫾ 7
307 ⫾ 8
304 ⫾ 5
308 ⫾ 7
390 ⫾ 17
391 ⫾ 22
375 ⫾ 20
372 ⫾ 10
426 ⫾ 21
420 ⫾ 29
398 ⫾ 25*
384 ⫾ 12**
432 ⫾ 21
426 ⫾ 25
403 ⫾ 25*
382 ⫾ 16**
336 ⫾ 104
293 ⫾ 85
324 ⫾ 116
350 ⫾ 129
321 ⫾ 70
328 ⫾ 49
283 ⫾ 57
281 ⫾ 62
388 ⫾ 95
301 ⫾ 119
234 ⫾ 75**
216 ⫾ 103**
341 ⫾ 136
292 ⫾ 114
210 ⫾ 123
174 ⫾ 94*
221 ⫾ 43
211 ⫾ 40
209 ⫾ 67
209 ⫾ 39
307 ⫾ 27
238 ⫾ 63**
228 ⫾ 44**
236 ⫾ 25**
289 ⫾ 56
281 ⫾ 65
238 ⫾ 49
143 ⫾ 91**
353 ⫾ 69
275 ⫾ 67
248 ⫾ 69*
156 ⫾ 74**
Note. Parentheses depict the number of rats. Values are the mean ⫾ standard deviations for each group. Asterisks indicate a significant difference from the
control (*p ⬍ 0.05, **p ⬍ 0.01, Dunnett’s comparison).
respectively. One rat in the control for evaluation of quantitative indices was excluded because it showed serious splenoma
with histopathological change of erythroblast hyperplasia in
red pulp. The blood biochemical data of another rat in the
200-ppm group were excluded because of hemolysis due to
mechanical injury during blood collection failure.
The rats of the 800-ppm group showed weak kicking on the
floor with their hind limbs, poor extension, and poor outspreading of pedal digits, turning up their planta when landing on the
plane (up-and-down landing). They could not stand still on the
slope, while the control rats could easily do so on the same
slope. Every rat of the 800-ppm group showed more or less
similar findings to those mentioned above. The rats of the 400or 200-ppm groups did not show clear abnormality in walking
status. Body weight gain was suppressed dose-dependently, but
there was no suppression at 200 ppm (Table 1). Grip strength
of forelimb and hind limb decreased dose-dependently and
progressively with exposure period (Table 1). Forelimb grip
strength decreased progressively at 400 ppm or more. Hind
limb grip strength significantly decreased at 200 ppm or more
after 4 weeks of exposure, but after 8 weeks of exposure, the
decrease was significant only in the 800-ppm group. After 12
weeks the decrease was significant only at 400 ppm or more.
MCV decreased and DL increased progressively at 800 ppm,
but not at 400 or 200 ppm (Table 2).
Light microscopic observation of the unraveled specimens
(Fig. 1) and epoxy resin-embedded sections (Fig. 2) of the
peripheral nerve showed ovoid or bubble-like debris of myelin
sheaths in the 800-ppm group, but not in the 400-ppm or
200-ppm group. Electron microscopic observation of the tibial
nerve of the 800-ppm group showed swelling of the axon, in
which we observed lamellar bodies, bubble-like vesicular condensation, amorphous material involving mitochondria, fine
filament tangles, and vesicular debris. Light-microscopic observation on the semi-thin epoxy resin sections of the gracile
nucleus showed light (Fig. 3, arrows) and dark (Fig. 3, arrowheads) preterminal swellings with thinned myelin. Under the
electron microscope, an accumulation of mitochondria (Fig.
4A), myelin-like debris (Fig. 4A), various sized vesicles (Fig.
4B), vacuolated mitochondria (Figs. 4B and 4C) and amorphous dense materials (Fig. 4C) were found. The light preterminal swelling profiles under light microscopy (Fig. 3, arrows)
seem to represent electron microscopic profiles with abundant
mitochondria (Fig. 4A) or vesicular debris (Fig. 4B), while the
dark preterminal swelling profiles (Fig. 3, arrowheads) show
accumulations of amorphous dense materials (Fig. 4C).
No degeneration was found in either gray matter or white
TABLE 2
Time Course of Maximum Motor Nerve Conduction Velocity
(MCV) and Distal Latency (DL) of Tail Nerve
Week
MCV (m/sec)
Control (8)
200 ppm (9)
400 ppm (9)
800 ppm (9)
DL (m/sec)
Control (8)
200 ppm (9)
400 ppm (9)
800 ppm (9)
0
24.9 ⫾ 1.9
24.6 ⫾ 2.1
24.2 ⫾ 1.8
24.5 ⫾ 1.6
2.9 ⫾ 0.2
2.9 ⫾ 0.2
2.9 ⫾ 0.2
3.0 ⫾ 0.2
4
28.8 ⫾ 4.6
29.8 ⫾ 3.7
30.2 ⫾ 3.0
28.0 ⫾ 4.0
2.5 ⫾ 0.2
2.8 ⫾ 0.3
2.8 ⫾ 0.4
3.1 ⫾ 0.5**
8
31.8 ⫾ 1.8
31.8 ⫾ 2.6
30.4 ⫾ 5.6
27.0 ⫾ 3.3*
2.7 ⫾ 0.2
2.8 ⫾ 0.2
2.8 ⫾ 0.1
3.4 ⫾ 0.4**
12
29.6 ⫾ 3.1
29.5 ⫾ 4.9
28.5 ⫾ 3.7
22.9 ⫾ 4.1**
2.8 ⫾ 0.3
2.7 ⫾ 0.2
3.0 ⫾ 0.3
4.3 ⫾ 0.8**
Note. Parentheses depict the number of rats. Values are the mean ⫾ standard
deviations for each group. Asterisks indicate a significant difference from the
control (*p ⬍ 0.05, **p ⬍ 0.01, Dunnett’s comparison).
119
NEUROTOXICITY OF 1-BROMOPROPANE
FIG. 1. Photomicrographs of unraveled muscular branch of posterior
tibial nerve. Control (A), no abnormality was found. 800-ppm group
(B), ovoid or bubble-like debris of
myelin sheath was found. Bar ⫽ 100
␮m; magnification of (A) and (B) is
the same.
matter at the examined transverse sections of the brain through
the optic chiasma, caudal margin of the mamillary body, or
caudal pole of the pons.
Irregular bandings of the striated muscle fibers were found in
the soleus muscle of the 800-ppm group under light microscopy (Fig. 5). Electron microscopic observations revealed a
loss of regular linearity in the Z line and zigzag arrangement of
the myofilaments (Fig. 6).
The weight of the cerebrum decreased significantly at 800
ppm compared to the control, whereas weights of the cerebellum and brainstem did not change significantly (Table 3).
Plasma CPK activity decreased dose-dependently, but the activity of LDH, ALP, ChE, GOT, and GPT enzymes did not
change (Table 4). Total cholesterol decreased dose-dependently, and total protein, albumin and globulin increased dosedependently. The former 3 parameters changed significantly at
FIG. 2. Photomicrograph of tibial nerve in epoxy resin-embedded, semithin section; 800-ppm group: ovoid or bubble-like debris of myelin was found.
Toluidine blue. Bar ⫽ 100 ␮m.
400 ppm and 800 ppm, but globulin changed significantly only
at 800 ppm. No other laboratory data showed any significant
changes.
DISCUSSION
All the rats in the exposed groups showed less severe debilitation and weight loss than the rats exposed to 1-bromopropane at 1000 ppm in our previous study (Yu et al., 1998),
which showed a great decrease in body weight after 4 –5
weeks. Walking status, such as up-and-down landing on a
plane and inability to stand still on a slope, showed the lack of
muscle strength of hind limbs in the 800-ppm group. This was
FIG. 3. Photomicrographs of preterminal axons in gracile nucleus. (A) and
(B) both from 800-ppm group. Swelling of preterminal axon with thinned
myelin sheath (arrows). Swelling of preterminal axon containing dark-stained
material (arrowheads). Toluidine blue. Bar ⫽ 50 ␮m; magnification of (A) and
(B) is the same.
120
ICHIHARA ET AL.
FIG. 4. Electron micrographs of preterminal axon in gracile nucleus (800-ppm group). (A) Swelling of axon with thinned myelin sheath. Accumulation of
mitochondria, myelin figures, and much debris were found; (B) swelling of axon with thinned myelin sheath. Various sized vesicles, vacuolated mitochondria,
and swelling granules were found. (C) Amorphous electron-dense materials were found in the central part of axon with vacuolated mitochondria around them.
Bar ⫽ 1 ␮m.
supported quantitatively by the dose-dependent decrease in the
grip strength of hind limbs, which indicated the strength of the
flexor musculature there.
Decrease in grip strength at 400 ppm and transiently at 200
ppm could not be explained by deterioration of MCV and DL
or morphological abnormalities, both of which were found
only at 800 ppm. Since the grip strength examination could
reflect total vital factors involved with the functions of limbs,
they might be more sensitive than morphological examination
or measurement of MCV or DL. Decrease in grip strength at
lower concentrations might be due to some factors detectable
only by the biochemical method. Plasma CPK activity de-
FIG. 5. Photomicrograph of longitudinal section of soleus muscle. Irregular bandings of muscle fibers were found in the 800-ppm group. Toulidine
blue. Bar ⫽ 100 ␮m.
creased specifically and dose-dependently with a significant
change at 400 ppm or more. This showed that plasma CPK
activity was more sensitive than MCV, DL, or histopathological signs. We could not determine whether the decrease in
CPK activity was involved with low muscle strength. However, the decrease in CPK activity might indicate some biochemical changes involved with toxic effects induced by 1-bromopropane or its metabolites. This may also be suggested by
the fact that administration of acrylamide, a neurotoxicant,
specifically decreased CPK activity in plasma or brain as well
as increasing landing-foot spread (Matsuoka et al., 1996).
The earlier change in DL than in MCV might indicate earlier
degeneration of myelin and axon in distal nerves. However, we
cannot disprove the possibility that it might indicate a delay in
the neuromuscular junction, since DL includes not only the
duration of distal nerve conduction but also that of chemical
transduction. A degenerative change of irregular banding of the
muscle fibers showed adverse effects of 1-bromopropane on
muscle fiber. However, it was unknown whether the degeneration was neurogenic or myogenic.
The morphological features of the degenerated peripheral
nerve were quite different from the axonal swelling, with
masses of neurofilament in a case of hexane intoxication
(Spencer and Schaumburg, 1977b), but rather similar to those
of Wallerian degeneration (Chaudhry et al., 1992; Griffin et al.,
1996; Johnson, 1997) or morphological change in rats administered acrylamide (Fullerton and Barnes, 1966; Schaumburg et
al., 1974).
The preterminal axonal swelling in the gracile nucleus was
also observed in rats exposed to hexane or acrylamide (Spencer
et al., 1977a), and, on fewer occasions, also in non-treated rats
(Lampert et al., 1964). Their common change was an accumu-
121
NEUROTOXICITY OF 1-BROMOPROPANE
FIG. 6. Electron micrographs of longitudinal section of soleus muscle. (A) 400-ppm group, low magnification: no abnormality was found. (B) 800-ppm
group, high magnification: I band showed irregularity. (C) 800-ppm group, high magnification: zigzag filamentous structures were found with glycogen particles
or mitochondria between them. Bar ⫽ 1 ␮m.
lation or increase of neurofilaments (Lampert et al., 1964;
Spencer et al., 1977a) under electron microscopy, although
accumulations of mitochondria (Lampert et al., 1964; Spencer
et al., 1977a), electron dense material (Lampert et al., 1964), or
other substances were also observed. The present experiment
did not show a predominant accumulation of neurofilaments,
but did reveal various degenerated materials. This might characterize 1-brompropane intoxication, but also might be possibly due to the difference in the level or part of the nerve, the
period of exposure, the concentration of chemicals, and so on.
Zhao et al. (1999) reported no difference between 1-bromopropane and 2-bromopropane in the effects of their subcutaneous injections on MCV or motor latency. However, the
present study and our preliminary study (Yu et al., 1998)
showed more potent neurotoxicity of 1-bromopropane, than
with 2-bromopropane (Yu et al., 1999a). In the present study,
1-bromopropane significantly decreased MCV at 800 ppm after
8 weeks (15.1%) and 12 weeks (22.4%) and increased DL after
4, 8, and 12 weeks (25.9%, 24.8%, and 51.4%, respectively,
compared to the control). On the other hand, exposure to
2-bromopropane, even at 1000 ppm, did not significantly decrease MCV after 12 weeks nor did it change DL after 4 weeks
(Yu et al., 1999a). It increased DL after 8 weeks and 12 weeks
at 1000 ppm, but the degree was almost equal to (26.4% after
8 weeks) or less than (36.7% after 12 weeks) exposure to
1-bromopropane at 800 ppm. 2-Bromoproane exposure did not
increase preterminal axonal swelling in the gracile nucleus, but
induced only ball or balloon-like swelling of the myelin sheath
TABLE 3
Weight of Cerebrum, Cerebellum, Brainstem, Soleus Muscle and Gastrocunemius Muscle
Number of rats
Brain
Cerebrum (g)
Cerebellum (g)
Brainstem (g)
Muscle
Soleus muscle (g)
Gastrocunemius muscle (g)
Control
200 ppm
400 ppm
800 ppm
8
9
9
9
1.14 ⫾ 0.03
0.28 ⫾ 0.01
0.64 ⫾ 0.04
1.13 ⫾ 0.03
0.28 ⫾ 0.01
0.62 ⫾ 0.02
1.11 ⫾ 0.03
0.27 ⫾ 0.02
0.63 ⫾ 0.04
1.05 ⫾ 0.04**
0.27 ⫾ 0.01
0.60 ⫾ 0.04
0.13 ⫾ 0.01
2.55 ⫾ 0.14
0.12 ⫾ 0.04
2.44 ⫾ 0.21
0.13 ⫾ 0.02
2.39 ⫾ 0.17
0.14 ⫾ 0.02
2.26 ⫾ 0.13**
Note. Values are the mean ⫾ standard deviations for each group. Asterisks indicate a significant difference from the control (**p ⬍ 0.01, Dunnett’s
comparison).
122
ICHIHARA ET AL.
TABLE 4
Effect of 1-Bromopropane Exposure on Enzymatic
Activity in Plasma
Number of rats
CPK (U/l)
ALP (U/l)
LDH (U/l)
GOT (U/l)
GPT (U/l)
Control
200 ppm
400 ppm
800 ppm
8
339 ⫾ 130
528 ⫾ 77
248 ⫾ 106
89 ⫾ 17
40 ⫾ 8
8
288 ⫾ 93
519 ⫾ 69
264 ⫾ 42
92 ⫾ 8
32 ⫾ 4
9
167 ⫾ 40**
484 ⫾ 96
289 ⫾ 168
119 ⫾ 50
34 ⫾ 13
9
113 ⫾ 25**
574 ⫾ 104
202 ⫾ 69
85 ⫾ 15
25 ⫾ 25
Note. Values are the mean ⫾ standard deviations for each group. Asterisks
indicate a significant difference from the control (**p ⬍ 0.01, Dunnett’s
comparison).
near Ranvier’s node in the peripheral nerve at 1000 ppm (Yu
et al., 1999a).
Ohnishi et al. (1999) exposed rats to 1-bromopropane at a
single concentration of 1500 ppm for 4 weeks and found ataxic
gait in rats. We could not define cerebellular ataxia in rats,
since the 4-footed walking rat was quite different from a
bipedal walking human in terms of cerebellular ataxia expression. Although pyknotic degeneration of cerebellular Purkinje
cells was found in the rats exposed to 1-bromopropane at 1000
ppm for 4 –5 weeks (Yu et al., 1998) and at 1500 ppm for 4
weeks (Ohnishi et al., 1999), the present study did not show
such change at 800 ppm and lower for 12 weeks.
In addition to the neurotoxicity of 1-bromopropane, we have
recently revealed its reproductive toxicity in male rats (Ichihara
et al., 1998b, 1999b, 2000).
In conclusion, 1-bromopropane weakened the muscle
strength of rat limbs and deteriorated MCV and DL, in a
concentration-dependent and exposure period-dependent manner. 1-Bromopropane was revealed to have a more potent
neurotoxicity than 2-bromopropane. Histopathological studies
showed ovoid or bubble-like debris of myelin in the peripheral
nerve, preterminal swelling in the gracile nucleus, and irregular
banding of muscle fibers in soleus muscle. 1-Bromopropane
should be used carefully in the workplace, because it may be
seriously neurotoxic to humans in addition to its reproductive
toxicity.
ACKNOWLEDGMENTS
This study was partly supported by Grants 10470106 and 11670367 from the
Ministry of Education, Science, Sports, and Culture, Japan.
REFERENCES
Chaudhry, V., Glass, J., and Griffin, J. (1992). Wallerian degeneration in
peripheral nerve disease. Neurol. Clin. 10, 613– 627.
Fullerton, P., and Barnes, J. (1966). Peripheral neuropathy in rats produced by
acrylamide. Br. J. Ind. Med. 23, 210 –221.
Griffin, J., George, E., and Chaudhry, V. (1996). Wallerian degeneration in
peripheral nerve disease. Bailliere’s Clin. Neurol. 5, 65–75.
Ichihara, G., Asaeda, N., Kumazawa, T., Tagawa, Y., Kamijima, M., Yu, X.,
Kondo, H., Nakajima, T., Kitoh, J., Yu, I., Moon, Y., Hisanaga, N., and
Takeuchi, Y. (1996). Testicular toxicity of 2-bromopropane. J. Occup.
Health 38, 205–206.
Ichihara, G., Asaeda,N., Kumazawa, T., Tagawa, Y., Kamijima, M., Yu, X.,
Kondo, H., Nakajima, T., Kitoh, J., Yu, I., Moon, Y., Hisanaga, N., and
Takeuchi, Y. (1997). Testicular and hematopoietic toxicity of 2-bromopropane, a substitute for ozone layer-depleting chlorofluorocarbons. J. Occup.
Health 39, 57– 63.
Ichihara, G., Ding, X., Yu, X., Wu, X., Kamijima, M., Peng, S., Jiang, X., and
Takeuchi, Y. (1999a). Occupational health survey on workers exposed to
2-bromopropane at low concentrations. Am. J. Ind. Med. 35, 523–531.
Ichihara G., Saito I., Kamijima M., Yu X., Shibata E., Toida M. and Takeuchi
Y. (1998a) Urinary 2,5-hexanedione increases with potentiation of neurotoxicity in chronic coexposure to n-hexane and methyl ethyl ketone. Int Arch
Occup Environ Health 71, 100 –104.
Ichihara, G., Yu, X., Eiji, S., Kitoh, J., Asaeda, N., Xie, Z., Wang, H., and
Takeuchi, Y. (1998b). Neurotoxicity of 1-bromopropane. Sangyo Eiseigaku
Zasshi 40, 414 (Japanese).
Ichihara, G., Yu, X., Kitoh, J., Asaeda, N., Kumazawa, T., Iwai, H., Shibata,
E., Yamada, T., Wang, H., Xie, Z., Maeda, K., Tsukamura, H., Takeuchi, Y.
(2000). Reproductive toxicity of 1-bromopropane, a newly introduced alternative to ozone layer depleting solvents, in male rats. Toxicol. Sci. 54,
416 – 423.
Ichihara, G., Yu, X., Kitoh, J., Shibata, E., Asaeda, N., Yamada, T., Wang, H.,
Xie, Z., Kumazawa, T., Iwai, K., Maeda, K., Tsukamura, H., and Takeuchi,
Y. (1999b). Histopathological changes of nervous system and reproductive
organ and blood biochemical findings in rats exposed to 1-bromopropane.
Sangyo Eiseigaku Zasshi 41, 513 (Japanese).
Ichihara, G., Yu, X., Kitoh, J., and Takeuchi, Y. (1998c). Neurotoxicity of
2-bromopropane and 1-bromopropane, substitutes for chlorofluorocarbons.
Peripheral Nerve 9, 69 –76 (Japanese).
Johnson, P. (1997). Peripheral nerve. In Textbook of Neuropathology. (L.
Davis and D. Robertson, Eds.), pp. 1233–1323. Williams & Wilkins, Baltimore.
Kamijima, M., Ichihara, G., Kitoh, J., Tsukamura, H., Maeda, K., Yu, X., Xie,
Z., Nakajima, T., Asaeda, N., Hisanaga, N., and Takeuchi, Y. (1997a).
Ovarian toxicity of 2-bromopropane in the non-pregnant female rat. J.
Occup. Health 39, 144 –149.
Kamijima, M., Ichihara, G., Kitoh, J., Tsukamura, H., Maeda, K., Yu, X., Xie,
Z., Nakajima, T., Asaeda, N., Hisanaga, N., and Takeuchi, Y. (1997b).
Disruption in ovarian cyclicity due to 2-bromopropane in the rat. J. Occup.
Health 39, 3– 4.
Kim, Y., Jung, K., Hwang, T., Jung, G., Kim, H., Park, J., Kim, J., Park, J.,
Park, D., Park, S., Choi, K., and Moon Y. (1996). Hematopoietic and
reproductive hazards of Korean electronic workers exposed to solvents
containing 2-bromopropane. Scand. J. Work Environ. Health 22, 387–391.
Lampert, P., Blumberg, J., and Pentschew, A. (1964). An electron microscopic
study of dystrophic axons in the gracile and cuneate nuclei of vitamin
E-deficient rats. J. Neuropathol. Exp. Neurol. 23, 60 –77.
Lim, C., Maeng, S., Lee, J., Chung, Y., Kim, T., Park, J., Moon, Y., and Yu,
I. (1997). Effects of 2-bromopropane on the female reproductive function in
Sprague-Dawley rats. Ind. Health 35, 278 –284.
Matsuoka, M., Matsumura, H., and Igisu, H. (1996). Creatine kinase activities
in brain and blood: Possible neurotoxic indicator of acrylamide intoxication.
Occup. Environ. Med. 53, 468 – 471.
Meyer, O., Tilson, H., Byrd, W., and Riley, M. (1979). A method for the
routine assessment of fore- and hind-limb grip strength of rats and mice.
Neurobehav. Toxicol. 1, 233–236.
NEUROTOXICITY OF 1-BROMOPROPANE
Nakajima, T., Shimodaira, S., Ichihara, G., Asaeda, N., Kumazawa, T., Iwai,
H., Ichikawa, I., Kamijima, M., Yu, X., Xie, Z., Kodo, H., and Takeuchi,Y.
(1997a). 2-Bromopropane-induced hypoplasia of bone marrow in male rats.
J. Occup. Health 39, 228 –233.
Nakajima, T., Shimodaira, S., Ichihara, G., Asaeda, N., Kumazawa, T., Iwai,
H., Ichikawa, I., Kamijima, M., Yu, X., Xie, Z., Kondo, H., and Takeuchi,
Y. (199b7). Histopathological findings of bone marrow induced by 2-bromopropane in male rats. J. Occup. Health 39, 81– 82.
Ohnishi, A., Ishidao, T., Kasai, T., Arashidani, K., and Hori, H. (1999).
Neurotoxicology of 1-bromopropane in rats. Sangyo Ika Daigaku Zasshi 21,
23–28. (Japanese)
Ono, Y., Takeuchi, Y., and Hisanaga, N. (1979). Studies on the methods of
measuring nerve conduction velocity in rat tails and on the comparative
toxicity of n-hexane, methyl n-butylketone and 2,5-hexanedione. Jpn. J. Ind.
Health 21, 528 –538.
Park, J., Kim, Y., Park, D., Choi, K., Park, S., and Moon, Y. (1997) An
outbreak of hematopoietic and reproductive disorders due to solvents containing 2-bromopropane in an electronic factory, South Korea: Epidemiological survey. J. Occup. Health 39, 138 –143.
Schaumburg, H., Wisniewski, H., and Spencer, P. (1974). Ultrastructural
studies of the dying-back process: I. J. Neuropathol. Exp. Neurol. 33,
260 –284.
Spencer, P., and Schaumburg, H. (1977a). Ultrastructural studies of the dyingback process: III. The evolution of experimental peripheral giant axonal
degeneration. J. Neuropathol. Exp. Neurol. 36, 276 –299.
Spencer, P., and Schaumburg, H. (1977b). Ultrastructural studies of the dyingback process: IV. Differential vulnerability of PNS and CNS fibers in
123
experimental, central-peripheral distal axonopathies. Neuropathol. Exp.
Neurol. 36, 300 –320.
Takeuchi, Y., Huang, J., Shibata, E., Hisanaga, N., and Ono, Y. (1989). A trial
to automatize an organic solvent exposure system for small animals. Jpn.
J. Industrial Health 31, 722 (Japanese).
Takeuchi, Y., Ono, Y., Hisanaga, N., Kitoh, J., and Sugiura, Y. (1980). A
comparative study on the neurotoxicity of n-pentane, n-hexane, and nheptane in the rat. Br. J. Ind. Med. 37, 241–247.
Yu, I., Chung, Y., Lim, C., Maeng, S., Lee, J., Kim, H., Lee, S., Kim, C., Kim,
T., Lim, C., Park, J., and Moon, Y. (1997) Reproductive toxicity of 2-bromopropane in Sprague Dawley rats. Scand. J. Work Environ. Health 23,
281–288.
Yu, X., Ichihara, G., Kitoh, J., Xie, Z., Asaeda, N., Shibata, N., Hisanaga, N.,
and Takeuchi, Y. (1999a). Effect of inhalation exposure to 2-bromopropane
on the nervous system in rats. Toxicology 135, 87–93.
Yu, X., Ichihara, G., Kitoh, J., Xie, Z., Shibata, E., Kamijima, M., Asaeda, N.,
and Takeuchi, Y. (1998). Preliminary report on the neurotoxicity of 1-bromopropane, an alternative solvent for chlorofluorocarbons. J. Occup. Health
40, 234 –235.
Yu, X., Kamijima, M., Ichihara, G., Li, W., Shibata, E., Hisanaga, N., and
Takeuchi, Y. (1999b). 2-Bromopropane causes ovarian dysfunction by damaging primordial follicles and their oocytes in female rats. Toxicol. Appl.
Pharmacol. 159, 185–193.
Zhao, W., Aoki, K., Xie, T., and Misumi, J. (1999). Electrophysiological
changes induced by different doses of 1-bromopropane and 2-bromopropane. J. Occup. Health 41, 1–7.