Biochemical Changes in the Central Nervous System of Rats Exposed

to 1-Bromopropane for Seven Days

Hailan Wang,*,1 Gaku Ichihara,* Hidenori Ito,† Kanefusa Kato,† Junzoh Kitoh,‡ Tetsuya Yamada,* Xiaozhong Yu,*

Seiji Tsuboi,§ Yoshinori Moriyama,§ Rie Sakatani,* Eiji Shibata,¶ Michihiro Kamijima,*

Seiichiro Itohara,* and Yasuhiro Takeuchi*

*Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya, Japan; †Department of Biochemistry,

Institute for Developmental Research, Aichi Human Service Center, Kasugai, Japan; ‡Nagoya University Graduate School of Medicine, Nagoya, Japan;

§Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Japan; and ¶Department of Medical Technology,

Nagoya University School of Health Sciences, Nagoya, Japan

Received November 12, 2001; accepted January 14, 2002

1-Bromopropane is used widely as an alternative to ozonedepleting solvents. The neurotoxic effects of this agent have been

described in humans and experimental animals. Here we investigated the underlying mechanisms of the neurotoxic effects of

1-bromopropane by examining the initial biochemical changes in

the central nervous system. Four groups of 9 Wistar male rats each

were exposed to 200, 400, or 800 ppm 1-bromopropane or only

fresh air, 8 h per day for 7 days. At the end of the experiment, the

cerebrum, cerebellum, brain stem and lumbar enlargement of the

spinal cord were dissected out from each rat (n 5 8) for biochemical analyses. Morphological examinations of the nervous system

were performed in the remaining rat of each group. 1-Bromopropane dose-dependently decreased neurospecific g-enolase, total

glutathione, and nonprotein sulfhydryl groups in the cerebrum

and cerebellum. Creatine kinase activity decreased dose-dependently in the brain and spinal cord. Histopathological examination

showed swelling of preterminal axons in gracile nucleus and degeneration of myelin in peripheral nerves. Our results of low levels

of g-enolase suggested that 1-bromopropane might primarily

cause functional or cellular loss of neurons in the cerebrum and

cerebellum. Glutathione depletion or modification to functional

proteins containing a sulfhydryl base as a critical site might be the

underlying mechanism of 1-bromopropane neurotoxicity.

Key Words: 1-bromopropane; creatine kinase; glutathione;

g-enolase; peripheral nerve; neurotoxicity.

1-Bromopropane is an important alternative to ozone-depleting solvents such as specific chlorofluorocarbons and 1,1,1-

trichloroethane. It is used as a cleaning agent for metals,

precision instruments, electronics, optical instruments, and ceramics. However, previous experimental studies from our laboratories have shown that 1-bromopropane has toxic effects on

various body systems such as the central nervous system and

reproductive system (Ichihara et al., 2000a,b; Yu et al., 1998).

Specifically, our study on the neurotoxicity of 1-bromopropane

showed decreased limb muscle strength, electrophysiological

and morphological changes in peripheral nerves, and swelling

of preterminal axons in the gracile nucleus of the medulla

oblongata. No other regions of the central nervous system

showed any clear morphological changes, although the weight

of the cerebrum was decreased. At about the time of reporting

our neurotoxicity study, a case was reported in the United

States of a 19-year-old male who presented with various neurological symptoms such as weakness of the lower extremities

and right hand, numbness, dysphagia, and urinary difficulties

following a 2-month exposure to an industrial solvent constituted mainly of 1-bromopropane (Sclar, 1999). Several tests in

that case showed specific abnormalities such as changes in

periventricular white matter and in areas close to the root

ganglia, as detected on magnetic resonance image (MRI), and

abnormal findings on somatosensory evoked potential studies

suggestive of lesions of the myelinated tracts in the central

nervous system.

The cerebrum weight loss in our rats and the MRI findings

in the reported patient suggested that 1-bromopropane had

adverse effects on the central nervous system. Accordingly, we

designed the present study to clarify the underlying mechanism

of the neurotoxic effects of 1-bromopropane by examining the

initial biochemical changes in the central nervous system.

For this purpose, we measured selected biochemical indices.

First, we measured neuron-specific g-enolase and glia-specific

b-S100 protein in the central nervous system to elucidate the

susceptible cells in each region. g-Enolase is localized in the

cytoplasm of neurons (Schmechel et al., 1978), and b-S100

protein is specifically distributed in glia cells (Cicero et al.,

1970; Isobe et al., 1990; Moore, 1975). These 2 markers are

useful in estimating neurological diseases (Royds et al., 1981;

Vassilopoulos and Jockers-Wretou, 1987) and solvent-related

neurotoxicity (Huang et al., 1989, 1990, 1992). Second, we

measured creatine kinase activity in the central nervous sys1 To whom correspondence should be addressed at Department of Occupational and Environmental Health, Nagoya University Graduate School of

Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Fax: (81)

(52) 744-2126. E-mail: wanghl@med.nagoya-u.ac.jp.

TOXICOLOGICAL SCIENCES 67, 114–120 (2002)

Copyright © 2002 by the Society of Toxicology

114
tem, based on reduced plasma creatine kinase (CK) activity

found in our previous 12-week experiment. We also measured

the activities of glutamic oxaloacetic transaminase (GOT) and

lactate dehydrogenase (LDH) to confirm whether change in

creatine kinase activity is specific to this enzyme. CK is present

in neurons, astrocytes, and oligodendrocytes (Manos et al.,

1991) and plays a role in continuous replenishment of ATP

from phosphocreatine in these cells (Wyss et al., 1992). Acrylamide and ethylene oxide, which were both neurotoxic, suppressed creatine kinase activity in the brain and blood of rats

(Kohriyama et al., 1994; Matsuoka et al., 1990, 1996). Moreover, we measured levels of creatine kinase isozymes creatine

kinase-BB (CK-B) and creatine kinase-MM (CK-M) in the

central nervous system and plasma using enzyme immunoassays to investigate whether decrease in CK activity is due to

enzymatic inhibition or decrease in enzyme amount. Third, we

measured the glutathione and sulfhydryl base of protein and

nonprotein fractions in the central nervous system. We hypothesized that nucleophilic reagents such as sulfhydryl base could

be a target of 1-bromopropane, because glutathione conjugates

1-bromopropane in rats (Barnsley et al., 1966; Jones and

Walsh, 1979).

In addition, we examined early histopathological changes in

the central nervous system and peripheral nerves to clarify the

relationship between these biochemical markers and biological

changes.

MATERIALS AND METHODS

Animals and exposure to 1-bromopropane. Thirty-six specific pathogenfree, 10-week-old (body weight 240–260 g) male Wistar rats were purchased

from Shizuoka Laboratory Animal Center, Japan. They were housed and

acclimatized to the new environment for 1 week, then divided at random into

4 groups of 9 each. They were housed in a room set on 12:12 light:dark cycle

(lights on at 0900 h and off at 2100 h), stable relative humidity (67–60%), and

constant temperature (23–25°C). Food and water were provided ad libitum.

The 4 groups of rats were exposed to 200, 400, or 800 ppm 1-bromopropane

or fresh air in inhalation chambers for8ha day, 7 days. Daily exposure

commenced at 1400 and was terminated at 2200 h. The inhalation exposure

system has been described previously (Ichihara et al., 1997; Takeuchi et al.,

1989). The vapor concentration in the chamber was measured every 10 s by gas

chromatography and digitally controlled to within 6 5% of the target concentration. After the 7-day exposure, the measured 1-bromopropane gas concentrations in the 3 chambers were 196 6 11, 395 6 8, and 798 6 16 ppm

(mean 6 SD). 1-Bromopropane (99.81% purity) was supplied by Tosoh Co.,

Ltd., Japan. Rat body weight was measured before exposure and after 1, 3, and

7 days of exposure. Japanese law concerning protection and control of animals,

the standards related to the care and management of experimental animals, and

the Guide for Animal Experimentation of the Nagoya University School of

Medicine were followed strictly throughout the experiment.

Organ weight and blood biochemical indices. After 7 days of exposure, 8

rats in each group were sacrificed by exsanguination through the abdominal

aorta under pentobarbital anesthesia. Plasma was separated by centrifugation at

room temperature and stored at –80°C until analysis. Plasma creatine kinase

activity and amount of CK-M isozyme were measured. The brain and spinal

cord (2 cm above the last thoracic vertebra, which corresponds with anterior

lumbar enlargement) were rapidly removed. The brain was immediately dissected into the cerebral hemisphere, cerebellum, and brain stem (medulla

oblongata, pons, and midbrain) on a steel plate placed on ice. Tissues of brain

and spinal cord were weighed and kept frozen at –80°C until analysis.

Immunoassays of nerve-specific marker proteins. Tissue blocks of the

brain and spinal cord were homogenized in 10 volumes (wt/vol) of 100 mM

citrate buffer (pH 7.4) containing 20 mM EDTA at 0°C. The homogenate was

centrifuged at 45,000 3 g for 20 min at 4°C. The supernatant was used for

analysis of nerve-specific proteins, activity of creatine kinase, GOT, LDH, and

for estimation of soluble protein concentrations. Neuronal marker protein

g-enolase, glial cell marker protein b-S100 protein, CK-B, and CK-M were

determined by the highly sensitive sandwich-type enzyme immunoassay system developed by Kato et al. (1981, 1982, 1986). Protein concentration of the

soluble fractions of homogenates was estimated by the dye binding method of

Bradford (1976) using Bio-Rad reagents (Bio-Rad, Richmond, CA).

Quantitative biochemical analyses. Protein-bound and nonprotein sulfhydryl groups were quantified essentially as described by Habeeb (1972). Tissues

were homogenized with 10 volumes (wt/vol) of 100 mM citrate buffer (pH 7.4)

containing 20 mM EDTA. Proteins were denatured with trichloroacetic acid at

a final concentration of 5% and pelleted by centrifugation at 15,000 3 g for 20

min. Each pellet was dissolved in 40 mM NaPB buffer (pH 8.0) containing 1%

SDS and 0.025% EDTA. Sulfhydryl groups in the pellet (protein-bound

sulfhydryl base, PSH) and the supernatant (nonprotein sulfhydryl base, NSH)

were quantified by incubation with 5,59-dithiobis-(2-nitrobenzoic; DTMB) in

80 mM NaPB buffer (pH 8.0) containing 2% SDS and 0.05% EDTA at room

temperature. For the quantification of total glutathione (total-GSH) and oxidized glutathione (GSSG), the supernatant was used and determined by the

method developed by Matsumoto et al. (1996). For the total-GSH determination, the supernatant was diluted 100 times with 125 mM sodium phosphate

(pH 7.5) containing 6.3 mM EDTA. An aliquot (50 ml) of the diluted solution

was assayed at 412 nm in a 1 ml mixture consisting of 0.21 mmol NADOH, 0.6

mmol DTMB, 125 mmol sodium phosphate (pH 7.5), and 6.3 mmol EDTA. For

GSSG determination, 20 ml of commercially available acrylonitrile (final

concentration, 295 mM), 50–200 ml of the supernatant and 125 mmol sodium

phosphate (pH 8.0) containing 6.3 mmol EDTA were mixed to give a final

volume of 1 ml, and were incubated at 25°C for 10 min. An aliquot of the

preincubation mixture was assayed.

Histopathological examination. The remaining rat of each group was

perfused from the left ventricle with Zamboni’s solution. Small tissue blocks

of cerebellum (posterior vermis), the gracile nucleus of the medulla oblongata,

thoracic spinal cord, dorsal root ganglion, and tibial nerve were embedded in

epoxy resin, cut into semi-thin sections, and stained by toluidine blue for light

microscopic examination. Segments of the posterior tibial nerve were dissected

out, postfixed in 0.5% osmium tetroxide, then immersed in 75% ethanol and

dehydrated in 50% glycerin. The nerve fibers were loosened and teased by

needles in 50% glycerin for light microscopic examination.

Statistical analysis. Data were expressed as mean 6 SD. Multiple comparisons between the exposure groups and the control were tested using

Dunnett’s method following one-way ANOVA. Significant statistical difference was set at p , 0.05.

RESULTS

Changes in Body and Brain Weights

Exposure to 800 ppm 1-bromopropane for 7 days resulted in

a significant loss of body weight compared with the control.

The weights of various brain regions did not change significantly following exposure to 1-bromopropane (Table 1).

Changes in Neuron-Specific Markers and Various Blood

Enzymes and Isozymes

Exposure of rats to 400 and 800 ppm 1-bromopropane

resulted in a significant decrease in tissue concentrations of

EXPOSURE TO 1-BROMOPROPANE 115
g-enolase in the cerebrum and cerebellum. No significant

changes were noted in b-S100 protein (Table 2).

The CK activity decreased dose-dependently in various regions of the brain and spinal cord in all exposed rats, but no

changes were noted in GOT and LDH levels (Table 3). CK-B

tissue concentrations decreased significantly in the cerebrum of

rats exposed to 800 ppm 1-bromopropane, however, it increased significantly in the brain stem of the same group, and

increased in the spinal cord in rats exposed to 400 and 800 ppm

1-bromopropane. No significant change was found in the

CK-M of brain by region or in that of spinal cord tissue (Table

4). On the other hand, plasma CK activity decreased dosedependently in the exposed groups, and the amount of CK-M

decreased dose-dependently in parallel with the enzymatic

activity (Table 5).

Changes in Total-GSH, GSSG, PSH, and NSH

Total glutathione concentrations were significantly lower in

the cerebrum and cerebellum of rats exposed to 800 ppm

compared to the control, but were significantly higher in the

spinal cord of all exposed rats. Furthermore, GSSG was lower

only in the brain stem of rats exposed to 400 ppm, and the

change was not concentration-dependent. NSH was significantly lower in both the cerebrum and cerebellum of 800 ppm

exposed rats, and in the brain stem of all exposed rats; however, it was higher in the spinal cord of rats exposed to 800

ppm. Exposure to 1-bromopropane did not alter the levels of

PSH (Table 6).

Histopathological Changes

Examination of epoxy resin-embedded sections of the gracile nucleus of rats exposed to 800 ppm 1-bromopropane

showed swelling of preterminal axon containing dark-stained

material with thin myelin sheath (Fig. 1). No significant change

was found in the sections of the cerebellum, dorsal root ganglion, or thoracic spinal cord.

Histopathological examination of the muscle branch of

the posterior tibial nerve, both in epoxy resin-embedded

section and segments that were loosened and teased by

needles, showed swelling or a dense mass of myelin sheath

especially near the nodes of Ranvier, hypertrophy of

Schwann cell cytoplasm and less frequency of SchmidtLanterman’s incisures in rats exposed to 800 ppm 1-bromopropane (Fig. 2).

TABLE 1

Body and Brain Weights in Rats after Exposure to 1-Bromopropane for 7 Days

Group Control 200 ppm 400 ppm 800 ppm

Body weight (g) 298 6 4.9 303 6 8.5 296 6 7.4 281 6 9.7*

Absolute organ weight

Whole brain (g) 1.86 6 0.03 1.86 6 0.05 1.87 6 0.04 1.82 6 0.04

Cerebrum (g) 1.05 6 0.02 1.07 6 0.03 1.06 6 0.03 1.04 6 0.02

Cerebellum (g) 0.27 6 0.01 0.27 6 0.01 0.27 6 0.01 0.27 6 0.01

Brain stem (g) 0.54 6 0.01 0.53 6 0.02 0.54 6 0.01 0.52 6 0.01

Note. Values are the means 6 SD for each group. For all groups, n 5 8.

*Significantly different from the control (p , 0.05, Dunnett’s multiple comparison).

TABLE 2

g-Enolase and b-S100 Protein in Rat Brain and Spinal Cord Tissues following 7 Days of 1-Bromopropane Exposure

Control 200 ppm 400 ppm 800 ppm

g-Enolase (ng/mg soluble protein)

Cerebrum 12.27 6 1.02 12.86 6 1.26 10.16 6 2.01* 9.67 6 1.60*

Cerebellum 20.13 6 4.59 15.37 6 3.48 14.43 6 2.90* 14.85 6 4.32*

Brain stem 9.80 6 1.09 9.88 6 1.51 10.04 6 1.28 9.72 6 1.21

Spinal cord 4.15 6 1.15 3.82 6 0.83 4.37 6 0.68 4.63 6 0.88

b-S100 (ng/mg souble protein)

Cerebrum 2.37 6 0.33 2.69 6 0.28 2.50 6 0.43 2.45 6 0.21

Cerebellum 3.97 6 1.08 4.20 6 0.62 4.12 6 0.93 3.94 6 0.59

Brain stem 3.72 6 0.36 3.74 6 0.47 3.96 6 0.49 3.98 6 0.58

Spinal cord 4.54 6 0.72 4.67 6 0.61 5.11 6 0.85 5.03 6 1.36

Note. Values are the means 6 SD for each group. For all groups, n 5 8.

*Significantly different from the control (p , 0.05, Dunnett’s multiple comparison).

116 WANG ET AL.
DISCUSSION

Our results showed that g-enolase and CK activity decreased

after exposure to 1-bromopropane at concentrations lower than

those at which morphological changes were initially detected

in the central nervous system. These results indicated that

biochemical indices are more vulnerable than morphological

structure to 1-bromopropane exposure, and might underlie the

mechanism of neurotoxic effects of 1-bromopropane. The

dose-dependent decrease in g-enolase might be specific to

1-bromoproane exposure, because exposure to toluene and

n-hexane does not reduce g-enolase in the rat brain; rather,

g-enolase and b-S100 increase following toluene exposure

(Huang et al., 1989, 1990, 1992). Since g-enolase is localized

specifically in neurons, a decrease in the concentration of this

enzyme might suggest a decrease in the amount of the enzyme

per cell or a decrease in the number of neurons. If the latter is

the case, it indicates a substantial toxic effect of 1-bromopropane on neurons.

In the present study, the inhibition of CK activities in the

brain and spinal cord seems to be the most sensitive indicator

of 1-bromopropane exposure. Based on the results of CK

activity and the amounts of CK-B and CK-M identified, we can

discuss whether the decrease in CK activity was due to the

decrease in CK amount. The amount of CK-M, which accounts

for 0.1–5% of total CK subunits in the central nervous system,

did not change significantly. The amount of CK-B, which

accounts for 99.9% of total CK subunits in the brain and 95%

in the spinal cord, did not parallel CK activity, and the decrease

in CK activity exceeded that of CK-B concentration in the

cerebrum. Furthermore, in the brainstem and spinal cord,

TABLE 3

CK, GOT, and LDH Activities in Rat Brain and Spinal Cord Tissues following 7 Days of 1-Bromopropane Exposure

Control 200 ppm 400 ppm 800 ppm

CK (IU/mg soluble protein)

Cerebrum 522.3 6 34.9 494.5 6 80.7 420.9 6 18.5* 324.2 6 23.9*

Cerebellum 737.0 6 158.6 707.6 6 65.4 680.4 6 58.5 539.2 6 33.7*

Brain stem 594.3 6 53.4 531.4 6 39.4* 471.1 6 32.4* 354.1 6 29.0*

Spinal cord 494.2 6 48.6 443.0 6 21.8* 391.0 6 36.2* 338.6 6 46.6*

GOT (IU/mg soluble protein)

Cerebrum 6.8 6 9.7 3.7 6 0.8 3.4 6 0.1 3.3 6 0.2

Cerebellum 5.6 6 1.3 5.2 6 0.3 5.0 6 0.2 4.8 6 0.4

Brain stem 5.1 6 0.5 5.1 6 0.3 5.2 6 0.4 5.0 6 0.3

Spinal cord 3.3 6 0.4 3.2 6 0.2 3.1 6 0.2 3.4 6 0.6

LDH (IU/mg soluble protein)

Cerebrum 6.8 6 0.3 7.2 6 1.7 6.6 6 0.4 6.4 6 0.4

Cerebellum 7.7 6 1.7 7.2 6 0.4 6.9 6 0.3 6.6 6 0.4

Brain stem 7.0 6 0.6 7.1 6 0.4 7.3 6 0.5 7.1 6 0.3

Spinal cord 4.9 6 0.5 4.7 6 0.3 4.7 6 0.3 5.0 6 0.8

Note. Values are the means 6 SD for each group. For all groups, n 5 8.

*Significantly different from the control (p , 0.05, Dunnett’s multiple comparison).

TABLE 4

CK-B and CK-M in Rat Brain and Spinal Cord Tissues following 7 Days of 1-Bromopropane Exposure

Control 200 ppm 400 ppm 800 ppm

CK-B (ng/mg soluble protein)

Cerebrum 17.75 6 2.15 16.26 6 1.55 16.36 6 1.14 14.30 6 1.76*

Cerebellum 24.59 6 4.91 23.73 6 1.47 22.43 6 1.80 22.60 6 2.49

Brain stem 16.16 6 1.29 15.69 6 2.55 18.37 6 1.21 19.25 6 2.50*

Spinal cord 12.34 6 0.98 13.54 6 1.11 14.78 6 2.29* 16.33 6 1.17*

CK-M (pg/mg soluble protein)

Cerebrum 11.99 6 7.26 13.51 6 9.40 10.57 6 4.89 9.92 6 5.99

Cerebellum 14.20 6 9.33 12.53 6 9.30 9.01 6 3.29 9.52 6 3.82

Brain stem 24.30 6 7.32 19.06 6 8.48 14.32 6 5.84 17.74 6 4.93

Spinal cord 522.93 6 233.63 651.65 6 170.74 349.12 6 98.62 430.93 6 294.32

Note. Values are the means 6 SD for each group. For all groups, n 5 8.

*Significantly different from the control (p , 0.05, Dunnett’s multiple comparison).

EXPOSURE TO 1-BROMOPROPANE 117
CK-B concentration did not decrease but rather increased,

although CK activity decreased after exposure to 1-bromopropane. Thus, the decrease in CK-B amount cannot fully explain

the fall in CK activity. It is possible that a considerable amount

of CK-B lost its enzymatic activity, suggesting enzymatic

inhibition probably through chemical modification of the enzyme. On the other hand, the plasma CK-M level almost

paralleled CK activity. It is also possible that only a small

proportion of CK-M that lost its activity was remaining in the

blood plasma, although it is also possible that the CK-M

antibody used in our study could not recognize CK-M denatured by 1-bromopropane exposure.

Our results also showed that exposure to 1-bromopropane

resulted in a significant decrease in tissue concentrations of

glutathione in the cerebrum and cerebellum, the site that also

showed reduced levels of g-enolase and CK activity. Glutathione depletion is thought to be associated with increased vulnerability of the brain to certain neurotoxic agents and contributes to oxidative damage of neurons and glial cells (Hu et al.,

1999; Trenga et al., 1991). Nonprotein SH levels almost correspond with those of GSH, and this was also valid under

exposure to 1-bromopropane. Glutathione has a SH-base,

which plays a role in reduction or conjugation of oxidative

agent or other toxic substances. CK also has a SH-base functional site, and the behavior of its enzymatic activity might

represent other functional proteins with SH-base (Zhou and

Tsou, 1987). It is possible that the neurotoxic effects of 1-bromopropane might include possible modification of functional

proteins containing a SH-base, represented by CK, in addition

to glutathione depletion. There was no increase in GSSG and

hence no evidence of oxidative stress. Our results showed

increased levels of total-GSH only in the spinal cord, contrary

to that in the cerebrum or cerebellum. It is possible that this

increase in total-GSH in the spinal cord represented a compensatory effect.

The exposure levels in the present study followed those in

TABLE 5

Plasma CK Activity and CK-M Content in Rats Exposed to 1-Bromopropane for 7 Days

Group Control 200 ppm 400 ppm 800 ppm

CK activity (IU/l) 464.7 6 113.4 438.0 6 147.1 345.1 6 124.4 173.7 6 49.3*

CK-M (ng/mg soluble protein) 3.38 6 2.08 3.15 6 1.87 1.94 6 1.00 1.23 6 1.17*

Note. Values are the means 6 SD for each group. For all groups, n 5 8.

*Significantly different from the control (p , 0.05, Dunnett’s multiple comparison).

TABLE 6

Total-GSH, GSSG, NSH, and PSH Levels in Rat Brain and Spinal Cord Tissues following 7 Days of 1-Bromopropane Exposure

Control 200 ppm 400 ppm 800 ppm

Total-GSH (nmol/g brain tissue)

Cerebrum 1031.9 6 156.9 1038.7 6 109.7 968.9 6 47.9 813.9 6 80.7*

Cerebellum 755.3 6 65.6 743.5 6 61.4 691.8 6 69.9 596.4 6 39.7*

Brain stem 613.8 6 34.0 682.4 6 65.0 640.8 6 92.6 602.9 6 100.8

Spinal cord 181.2 6 34.9 279.6 6 31.0* 333.9 6 38.5* 371.0 6 69.5*

GSSG (nmol/g brain tissue)

Cerebrum 18.7 6 1.1 19.8 6 3.0 20.3 6 2.3 19.7 6 2.3

Cerebellum 11.8 6 2.6 15.1 6 5.6 10.7 6 3.7 12.6 6 3.2

Brain stem 18.5 6 3.4 17.1 6 3.6 12.8 6 2.98 14.5 6 3.4

Spinal cord 17.1 6 3.4 18.7 6 2.9 19.9 6 3.8 20.4 6 2.1

NSH (umol/g brain tissue)

Cerebrum 1.567 6 0.094 1.520 6 0.105 1.472 6 0.056 1.276 6 0.067*

Cerebellum 1.191 6 0.082 1.133 6 0.081 1.171 6 0.146 0.946 6 0.093*

Brain stem 1.403 6 0.149 1.149 6 0.083 1.154 6 0.178* 1.082 6 0.020*

Spinal cord 0.606 6 0.080 0.653 6 0.052 0.707 6 0.105 0.741 6 0.105*

PSH (umol/g soluble protein)

Cerebrum 105.49 6 24.06 99.47 6 17.59 88.24 6 7.17 104.43 6 22.00

Cerebellum 172.17 6 39.29 164.56 6 16.04 149.48 6 20.02 146.14 6 20.66

Brain stem 136.43 6 16.12 130.87 6 3.17 136.01 6 11.34 130.69 6 4.61

Spinal cord 98.43 6 9.39 97.56 6 9.99 102.83 6 3.69 99.91 6 9.77

Note. Values are the means 6 SD for each group. For all groups, n 5 8.

*Significantly different from the control (p , 0.05, Dunnett’s multiple comparison).

118 WANG ET AL.
our previous 12-week study (Ichihara et al., 2000a,b). It was

reported that some workers were exposed to 1-bromopropane

at 18–381 ppm (mean 5 142) in the plants where 1-bromopropane was used as a solvent of spray adhesive without sufficient

ventilation or containment (NIOSH, 1999, 2000). One case

report with neurological disorders showed exposure levels

ranging from 60–261 ppm (mean 5 133 ppm; Ichihara et al.,

2002). The exposure levels in the present study covered the

range of the real exposure levels of workers using 1-bromopropane under worse conditions as seen in the above cases. However, it should be also noted that it might be difficult to

extrapolate from the present data to humans at this moment,

because the possibility of metabolic enhancement or species

difference of susceptibility has not been clarified yet.

In conclusion, we demonstrated in the present study that

1-bromopropane induced dose-dependently a decrease in neurospecific g-enolase in the cerebrum and cerebellum that suggested functional or cellular loss of neurons. This was accompanied by decreases in sulfhydryl base, total glutathione, and

creatine kinase activity. Glutathione depletion or modification

of functional proteins containing a sulfhydryl base might be the

underlying mechanism of 1-bromopropane-induced neurotoxFIG. 1. Photomicrographs of preterminal axons in gracile nucleus of rats. (A) Control, no abnormal changes. (B) Rat of exposed to 1-bromopropane 800

ppm. Note the swelling of the preterminal axon containing dark-stained material (arrowhead) and the thin myelin sheath (arrows). Toluidine blue. Magnification

of (A) and (B) is the same.

FIG. 2. Photomicrographs of the muscular branch of the posterior tibial nerve. (A) Control, no abnormal changes, Schmidt-Lanterman’s incisures (E) can

be seen clearly. (B) A representative rat exposed to 800 ppm 1-bromopropane. Note swelling or dense mass of myelin sheath (S) especially near the nodes of

Ranvier, and hypertrophy of Schwann cell cytoplasm (H). Magnification of (A) and (B) is the same.

EXPOSURE TO 1-BROMOPROPANE 119
icity. Our results also showed that the medulla oblongata and

peripheral nerves start to show morphological changes within

7 days of exposure.

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