* 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
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ABSTRACT |
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Key Words: 1-bromopropane; creatine kinase; glutathione; -enolase; peripheral nerve; neurotoxicity.
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INTRODUCTION |
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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 -enolase and glia-specific ß-S100 protein in the central nervous system to elucidate the susceptible cells in each region.
-Enolase is localized in the cytoplasm of neurons (Schmechel et al., 1978
), and ß-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 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., 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.
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MATERIALS AND METHODS |
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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., 1997; Takeuchi et al., 1989
). 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 -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), 50200 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.
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RESULTS |
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DISCUSSION |
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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 0.15% 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 -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 our previous 12-week study (Ichihara et al., 2000a,b
). It was reported that some workers were exposed to 1-bromopropane at 18381 ppm (mean = 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 60261 ppm (mean = 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 -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.
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NOTES |
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