Biochemical Changes in the Central Nervous System of Rats Exposed to 1-Bromopropane for Seven Days

Hailan Wang*,1, Gaku Ichihara*, Hidenori Ito{dagger}, Kanefusa Kato{dagger}, Junzoh Kitoh{ddagger}, 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; {dagger} Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Japan; {ddagger} 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1-Bromopropane is used widely as an alternative to ozone-depleting 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 = 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 {gamma}-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 {gamma}-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; {gamma}-enolase; peripheral nerve; neurotoxicity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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., 2000aGo,bGo; Yu et al., 1998Go). 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, 1999Go). 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 {gamma}-enolase and glia-specific ß-S100 protein in the central nervous system to elucidate the susceptible cells in each region. {gamma}-Enolase is localized in the cytoplasm of neurons (Schmechel et al., 1978Go), and ß-S100 protein is specifically distributed in glia cells (Cicero et al., 1970Go; Isobe et al., 1990Go; Moore, 1975Go). These 2 markers are useful in estimating neurological diseases (Royds et al., 1981Go; Vassilopoulos and Jockers-Wretou, 1987Go) and solvent-related neurotoxicity (Huang et al., 1989Go, 1990Go, 1992Go). Second, we measured creatine kinase activity in the central nervous system, 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., 1991Go) and plays a role in continuous replenishment of ATP from phosphocreatine in these cells (Wyss et al., 1992Go). Acrylamide and ethylene oxide, which were both neurotoxic, suppressed creatine kinase activity in the brain and blood of rats (Kohriyama et al., 1994Go; Matsuoka et al., 1990Go, 1996Go). 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., 1966Go; Jones and Walsh, 1979Go).

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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and exposure to 1-bromopropane.
Thirty-six specific pathogen-free, 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 for 8 h a 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., 1997Go; Takeuchi et al., 1989Go). The vapor concentration in the chamber was measured every 10 s by gas chromatography and digitally controlled to within ± 5% of the target concentration. After the 7-day exposure, the measured 1-bromopropane gas concentrations in the 3 chambers were 196 ± 11, 395 ± 8, and 798 ± 16 ppm (mean ± 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 x 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 {gamma}-enolase, glial cell marker protein ß-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 x 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 5,5`-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 µl) of the diluted solution was assayed at 412 nm in a 1 ml mixture consisting of 0.21 µmol NADOH, 0.6 µmol DTMB, 125 µmol sodium phosphate (pH 7.5), and 6.3 µmol EDTA. For GSSG determination, 20 µl of commercially available acrylonitrile (final concentration, 295 mM), 50–200 µl of the supernatant and 125 µmol sodium phosphate (pH 8.0) containing 6.3 µmol 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 ± 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
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 1Go).


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TABLE 1 Body and Brain Weights in Rats after Exposure to 1-Bromopropane for 7 Days
 
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 {gamma}-enolase in the cerebrum and cerebellum. No significant changes were noted in ß-S100 protein (Table 2Go).


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TABLE 2 {gamma}-Enolase and ß-S100 Protein in Rat Brain and Spinal Cord Tissues following 7 Days of 1-Bromopropane Exposure
 
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 3Go). 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 4Go). On the other hand, plasma CK activity decreased dose-dependently in the exposed groups, and the amount of CK-M decreased dose-dependently in parallel with the enzymatic activity (Table 5Go).


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TABLE 3 CK, GOT, and LDH Activities in Rat Brain and Spinal Cord Tissues following 7 Days of 1-Bromopropane Exposure
 

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TABLE 4 CK-B and CK-M in Rat Brain and Spinal Cord Tissues following 7 Days of 1-Bromopropane
 

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TABLE 5 Plasma CK Activity and CK-M Content in Rats Exposed to 1-Bromopropane for 7 Days
 
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 6Go).


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TABLE 6 Total-GSH, GSSG, NSH, and PSH Levels in Rat Brain and Spinal Cord Tissues following 7 Days of 1-Bromopropane Exposure
 
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. 1Go). No significant change was found in the sections of the cerebellum, dorsal root ganglion, or thoracic spinal cord.



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FIG. 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.

 
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 Schmidt-Lanterman's incisures in rats exposed to 800 ppm 1-bromopropane (Fig. 2Go).



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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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our results showed that {gamma}-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 {gamma}-enolase might be specific to 1-bromoproane exposure, because exposure to toluene and n-hexane does not reduce {gamma}-enolase in the rat brain; rather, {gamma}-enolase and ß-S100 increase following toluene exposure (Huang et al., 1989Go, 1990Go, 1992Go). Since {gamma}-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, 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 {gamma}-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., 1999Go; Trenga et al., 1991Go). 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, 1987Go). 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 our previous 12-week study (Ichihara et al., 2000aGo,bGo). It was reported that some workers were exposed to 1-bromopropane at 18–381 ppm (mean = 142) in the plants where 1-bromopropane was used as a solvent of spray adhesive without sufficient ventilation or containment (NIOSH, 1999Go, 2000Go). One case report with neurological disorders showed exposure levels ranging from 60–261 ppm (mean = 133 ppm; Ichihara et al., 2002Go). 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 {gamma}-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 neurotoxicity. Our results also showed that the medulla oblongata and peripheral nerves start to show morphological changes within 7 days of exposure.


    NOTES
 
1 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{at}med.nagoya-u.ac.jp. Back


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