# Introduction

2-Bromopropane (2BP) was introduced in to the semiconductor and other industries as a replacement for ozonedepleting chlorofluorocarbons. In 1995, Korean workers exposed to 2BP in an electronics factory suffered severe reproductive and hematopoietic disorders 1) 

# . Male workers developed oligozoospermia or azoospermia, and females developed amenorrhea, while pancytopenia was observed in both sexes, though the frequency ofhematopoietic disorders was lower than that of reproductive disorders. Therefore, the effects on reproductive

function were considered to be the most serious toxic effect of 2BP in humans. The reproductive toxicity of 2BP has been confirmed by animal experiments. Sperm counts are reduced in male rats injected with 2BP or exposed to 2BP gas 2) 

# . Exposure to 2BP disturbs theestrous cycle and elicits a decrease in the number of ovulated ova in female mice and rats 3–5) 

# . The reproductive and hematopoietic toxicity of 2BP has led to a decrease in its use and its replacement with 1-bromopropane (1BP). However, the results of animal experiments indicate that 1BP is toxic to the peripheral nerves 6, 7) 

# . The grip strength of all four limbs of male Wistar rats decreased following exposure to 800 ppm of 1BP for 8 h per day for 8 wk. Maximum motor nerve conduction velocity decreased and distal latency increased in the tail nerve at 800 ppm. Histopathological changes were evident in Industrial Health 2008, 46 , 348–359 

# Original Article



# Alteration of Brain Levels of Neurotransmitters and Amino Acids in Male F344 Rats Induced by Three-week Repeated Inhalation Exposure to 1-Bromopropane

Megumi SUDA 1 

# *, Takeshi HONMA 2 

# , Muneyuki MIYAGAWA 1 

# and Rui-Sheng WANG 1

1 

## National Institute of Occupational Safety and Health (JNIOSH), Nagao 6–21–1, Tama-ku, Kawasaki 214-

8585, Japan

2 

## Japan Association for Working Environment Measurement, Shiba 4–4–5, Minato-ku, Tokyo 108-8372, Japan

Received October 10, 2007 and accepted April 16, 2008

Abstract: The present study investigated the effects of 1-bromopropane (1BP) on brain neuroactive substances of rats to determine the extent of its toxicity to the central nervous system (CNS). We measured the changes in neurotransmitters (acetylcholine, catecholamine, serotoninand amino acids) and their metabolites or precursors in eight brain regions after inhalation exposure to 1BP at 50 to 1,000 ppm for 8 h per day for 7 d per week for 3 wk. Rats were sacrificedat 2 h (Case 1), or at 19 h (Case 2) after the end of exposure. In Case 1, the level of 5-hydroxyindoleacetic acid (5HIAA) was lowered in some brain regions by 1BP exposure. The decrease of 5HIAA in the frontal cortex was statistically significant at 50 ppm 1BP exposure. In Case 2,

γ

-amino butyric acid (GABA) and taurine were decreased in many brain regions of exposed rats, and a significant decrease of taurine in the midbrain occurred at 50 ppm 1BP exposure. In both cases of 2-h and 19-h intervals from the end of exposure to sacrifice, aspartate and glutamine levels were elevated in many brain regions, but the acetylcholine level did not change in any brain region. Three-week repeated exposure to 1BP produced significantly changes in amino acid contents of rat brains, particularly at 1,000 ppm.

Key words: 1-Bromopropane, Toxicity, Rat brain, Neurotransmitter, Acetylcholine, Catecholamine,

Serotonin, Amino acids

*To whom correspondence should be addressed.

# peripheral nerves and muscle fibers. However, the effects of 1BP on the central nervous system (CNS) have been poorly characterized. Histopathological studies showed that 1BP had no influence on the central and peripheral

nerves in rats 8) 

# , whereas some enzyme activities were influenced by exposure to 1BP 9, 10) 

# . Electrophysiologicalstudies demonstrated an inhibitory restraint in hippocampal cells in 1BP-exposed rats 10, 11) 

# . Our previous study, which was performed to investigate the effects of 1BP on CNS, showed behavioral excitation in rats following exposure to 1BP 12) 

# . The present study was undertaken todetermine the extent of CNS toxicity of 1BP by investigating the changes in neuroactive molecules in the brain.We measured the neurotransmitters (acetylcholine, catecholamine, serotonin, and amino acids) and their metabolites or precursors in rat brains after inhalation exposure to 1BP for 8 h per day for 7 d per week for 3 wk. In the reports cited above, animal exposure to 1BP was performed at 200 ppm or higher levels. However, in theworkplace, reported levels of human occupational exposure were at 60–261 ppm 13) 

# or 41.3–143 ppm 14) 

# . In our study, the level of 50 ppm was adopted to mimic the reported human exposures to 1BP.



# Materials and Methods

Animals

Male 8-wk-old F344 rats obtained from Charles River Japan Inc. were acclimatized in stainless steel wire net cages in groups of five per cage for at least 5 wk with a light/dark cycle of 12 h: 12 h (lights on at 8:00 h). The temperature and humidity in the breeding and exposurerooms were maintained at 23 ± 1 ̊C and 55 ± 5%, respectively. Food (CE-2, Japan Clea Inc.) and water were

accessible ad libitum

.

Experimental design and chemicals After acclimatization, the rats were grouped such that mean body weight did not differ among the groups. The rats inhaled vaporized 1BP (99%, Sigma-Aldrich Japan) in stainless steel chambers (Sibata Scientific Technology Ltd., Japan) as described previously 5, 12) 

# . The exposure concentrations of 1BP, 50, 200, and 1,000 ppm (251, 1,006, and 5,028 mg/m 3 

# , respectively), were monitored using a gas chromatograph (Shimadzu GC-7A, Japan) every 20 min and adjusted with flowmeters to a constant target value ± 5% throughout the exposure. Control ratswere exposed to clean air. We set up two cases for exposure-sacrifice intervals. In Case 1, rats were exposed to 1BP for 8 h every day for 7 d per week for 3 wk between 0:00 and 8:00 h, and were sacrificed at 2 h after the end of exposure. In Case 2, the exposure took place between 10:00 and 18:00 h, and the interval between the end of exposure and the sacrifice was 19 h. We chose 2 h as the exposure-sacrifice interval in Case 1 so that the results of Case 1 in the present study could be compared with those of our previous study concerning 1BP toxicity 12)



# .The interval of 19 h from the end of exposure to sacrifice in Case 2 was selected in order to observe the timecourse changes of 1BP effects. Exposure concentrations up to 1,000 ppm in the present study were chosen becausewe exposed rats to 1,000 ppm of 1BP and 2BP at maximum for 3 wk in our previous investigations performed to elucidate reproductive and CNS toxicity 12) 

# . In Cases 1 and 2, all experimental conditions were the same exceptthe exposure time, dark period in Case 1 and light period in Case 2, and the intervals from the end of exposure

to sacrifice. The experiments were conducted under the

“Institutional Guidelines for Animal Care and Use” of the National Institute of Occupational Safety and Health, Japan (Kawasaki).

Extraction of brain substances Rats were sacrificed by microwave exposure (4.7 KW, 1.5 s) focused on the head (Microwave applicator, Muromachi Kikai Co., Tokyo) 15) 

# . Exposure to microwaves rapidly increases the brain temperature and prevents rapid postmortem changes of brain substances. The brains were dissected into eight brain regions as described by

Glowinski and Iversen 16) 

# : the frontal cortex, occipital cortex, hippocampus, striatum, midbrain, hypothalamus, medulla oblongata, and cerebellum.

All brain samples were stored at –80 ̊C until analysis

and dissolved in 0.2 N HClO 4 

# containing 1 mM EDTA

and 5 mM Na 2 

# S 2 

# O 5 

# before disruption using a PotterElvehjem homogenizer. The homogenate was centrifuged at 12,000 × g for 25 min at 4 ̊C, and the supernatant was used for analysis 17, 18)



# .

Analysis of acetylcholine, choline, and amino acids Supernatants were divided into two portions (Portions A & B). Portion A was neutralized with potassium acetate and the supernatant was obtained after centrifugation. This supernatant was analyzed by HPLC to quantitate the acetylcholine (ACh) and amino acid contents.

ACh was measured following a procedure previously

described 19) 

# except for the HPLC pump, EP-300 (Eicom, Japan), and electrochemical detector, ECD-300 (Eicom). Amino acids in the supernatant of neutralized portionA were separated by high performance liquid chromatography using an ion exchange column (ion exchange resin #2619-F, 4 mm I.D. × 150 mm, Hitachi Co., Japan).Amino acid components were eluted by a gradient elution method, and were changed to fluorescent derivatives with o-phthalaldehyde. The fluorescence intensity was 1-BROMOPROPANE AND BRAIN NEUROTRANSMITTERS IN MALE RATS 

# 349

# measured using a fluorescence detector (F-1080, Hitachi). Mobile phases comprised 0.2 M lithium citrate buffer, atpH 3 and pH 7. The flow rate in the ion exchange column was 0.4 ml/min. The column temperature was maintained at 37 ̊C in a column oven. The detection wavelengths used were 340 nm for excitation and 450 nm for

emission.

Analysis of monoamines and their metabolites In Case 1 (sacrifice at 2 h after exposure), Portion Bwas applied to activated alumina to adsorb norepinephrine (NE), dopamine (DA), and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC). These amines and the metabolite were desorbed from alumina (Portion B1). Serotonin (5HT), and 5-hydroxyindoleacetic acid(5HIAA) remained in the eluate. The supernatant containing 5HT and 5HIAA was obtained by centrifugation (Portion B2). Each portion was passed through a filter

of 0.45 μ m pore size before performing HPLC. Monoamines and metabolites were assayed following a procedure previously described 19, 20)



# . In Case 2 (sacrifice at 19 h after exposure), Portion B was injected directly to HPLC equipped with a CoulArray ® 

# detector (ESA Inc., U.S.A.) after passing through a filter of 0.45 μ m pore size for analysis of NE, 3-methoxy-4-hydroxyphenylglycol (MHPG), DA, DOPAC, homovanillic acid (HVA), 5HT, and 5HIAA. Monoamines and metabolites were separated by reverse phase ion pair chromatography (MCM Column, 4.6mm

I.D. × 150mm, MC Medical, Inc., Japan) using a mobile phase comprised of 0.1 M phosphate-0.05 M citrate buffer, pH 3.0. This buffer contained sodium 1-heptanesulfonate,

EDTA · Na 2 

# · H 2 

# O, acetonitrile and methanol. The column temperature was maintained at 23 ̊C in an oven, and the flow rate was 1.1 ml/min. The voltages for CoulArray ® 

# detection were 100, 200, 300 and 400 mV.

Statistics

Means ± SD of each group were calculated from

monoamine or metabolite contents of each brain area (nmoles/g tissue) in each rat. Ratios for metabolite/ monoamine (MHPG/NE, DOPAC/DA, HVA/DA,5HIAA/5HT) were obtained in each rat and the mean values of these ratios were calculated in each group. Metabolite/monoamine ratios are widely used as markers of turnover of NE, DA and 5HT in cerebral neurons. Free amino acid contents of the brain areas ( μ moles/g tissue)

were obtained and the mean values were calculated in each group. The statistical significance of differences between the control and exposed groups was examined by Dunnett’s multiple t -test using statistics software (SPSS Japan Inc.). Differences between groups at p <0.05 were considered significant. 

# Results

Effects of 1BP on the brain substances in Case 1: Figure 1 shows the levels of ACh, DA, DOPAC, and DOPAC/DA ratio in the eight brain regions of the male rats at 2 h after the end of 3 wk inhalation exposure to 1BP. Brain contents of DA in the striatum at 50 ppmand DOPAC in the hippocampus at 1,000 ppm were significantly less than control values. Levels of NE, 5HT, 5HIAA, and the 5HIAA/5HT ratio are shown in Fig. 2. In the frontal cortex, the levels of 5HIAA in 1BP-treated groups were lowered and the differences from the control were significant at 50 and 1,000 ppm. In the striatum, the levels of 5HIAA were lowered in 1BP-treated groups in a dose-dependent manner, with statistical significance at 200 and 1,000 ppm 1BP exposure.

Figure 3 shows the levels of amino acids in three brain regions of rats at 2 h after the end of 3 wk inhalation exposure to 1BP. The sample volume was sufficient to quantitate amino acids only in three brain regions, because the measurement of ACh was given priority in sample Portion A. In the rat group exposed to 1,000 ppm 1BP, levels of aspartate and glutamine were higher than those of the control with statistical significance ( p <0.01). Such

increases in these two amino acids were not observed at 50 and 200 ppm 1BP exposure. The levels of γ -aminobutyric acid (GABA) and taurine (an amino acid containing sulfur) in the 1,000 ppm 1BP exposure group were lower than the control group, but the difference was only statistically significant for GABA in the hippocampus.

Effects of 1BP on the brain substances in Case 2: Figure 4 shows the levels of ACh, DA, DOPAC, and HVA, and the ratios of DOPAC/DA and HVA/DA in the eight brain regions of the rats at 19 h after the end of 3 wk inhalation exposure to 1BP. The levels of NE, MHPG, 5HT, and 5HIAA, and the ratios of MHPG/NE and 5HIAA/5HT are given in Fig. 5. In the 1BP-exposedgroups, the HVA level and the HVA/DA ratio in the striatum and the NE level in the hypothalamus decreased dosedependently, and differences from the control for the two substances were significant at 1,000 ppm. The MHPG level and the MHPG/NE ratio in the occipital cortex at 1,000 ppm 1BP exposure were significantly lower thanthose of the control. Levels of 5HT in the occipital cortex and 5HIAA in the medulla oblongata were significantly higher than those of the control.

Figure 6 shows the levels of amino acids in the eight brain regions of the rats at 19 h after the end of 3 wk inhalation exposure to 1BP. In the group exposed to 1BP at 1,000 ppm, the levels of aspartate were significantlyhigher than the control in the occipital cortex, hippocampus, striatum, hypothalamus, and cerebellum. Also, the 

# 350 M SUDA et al

.

Industrial Health 2008, 46 , 348–359

# levels of glutamine were significantly higher than the control in all brain regions except the medulla oblongata. The midbrain contents of glutamate were significantly reduced in the 50 and 200 ppm 1BP exposure groups, however,the decreases were small in degree and not dose-dependent. The levels of GABA in the 1,000 ppm 1BP exposure group were significantly lower than the control in the frontal and occipital cortex, and hippocampus. In the 1,000 ppm 1BP exposure group, the level of taurine was significantly lower than the control in all brain regions except the frontal cortex, the levels of taurine were also lowered by 1BP exposure at 50 ppm.

Table 1 shows the levels of free amino acids other thanthose shown in Fig. 6 in Case 2. The increases in cystathionine, serine, threonine, and β-alanine were significant in many brain regions, particularly at exposure to 1,000 ppm 1BP.



# Discussion

In both Cases 1 and 2, 1BP exposure elicited no effect on ACh contents in any of the eight brain regions. NE content in the hypothalamus, a brain region rich in NE,

was decreased and the MHPG level and MHPG/NE ratio was lowered in the occipital cortex in Case 2. These results suggest that 1BP exposure lowers activity in NE neurons within some brain regions.Regarding DA and its metabolite, significant differ1-BROMOPROPANE AND BRAIN NEUROTRANSMITTERS IN MALE RATS 

# 351

Fig. 1. Effects of 1BP exposure on brain levels of ACh, DA and DOPAC, and the DOPAC/DA ratio in male rats at 2 h after the end o f

3 wk inhalation exposure. The amounts of amines and metabolites are expressed in nmoles/g tissue. Results are shown as means ± SD (n=4–5).

*: p <0.05 by Dunnett’s multiple t -test.

# ences from control values were decreases in striatal DA at 50 ppm 1BP exposure and hippocampal DOPAC at 1,000 ppm 1BP exposure in Case 1. The DOPAC/DAratio tended lower, dose-dependently, although no statistical significance was found. In Case 2, HVA content and HVA/DA ratio in the striatum were significantly lower than the control at 1,000 ppm 1BP exposure. 1BP exposure probably lowered the activity of DA neurons, particularly in the striatum, a DA-rich brain region. Effects of 1BP exposure on 5HT and its metabolite, 5HIAA, were complicated. In Case 1, 5HIAA contents

of the frontal cortex and the striatum were reduced in 1BPexposure groups. On the other hand, 5HT in the occipital cortex and 5HIAA in the medulla oblongata increased in Case 2. These differences may be due to the time of 1BP exposure. In Case 1 rats received exposure in the dark period, while in Case 2 they were exposed to 1BP in the light period. It is well-known that 5HT neurons are deeply involved in sleep and circadian rhythm 21) 

# , and the situation of 5HT neurons in the dark period is quite different from that in the light period. 1BP exposure inthe dark period seemed to lower the activity of 5HT neurons, while 1BP exposure in the light period seemed to

enhance it.

In Case 1, GABA in the hippocampus decreased with statistical significance at high 1BP exposure. In Case 2, GABA contents decreased slightly but significantly in the frontal and occipital cortex and hippocampus. Fueta et 

# 352 M SUDA et al

.

Industrial Health 2008, 46 , 348–359 Fig. 2. Effects of 1BP exposure on brain levels of NE, 5HT and 5HIAA, and the 5HIAA/5HT ratio in male rats at 2 h after the end of

3 wk inhalation exposure. See legend for Fig. 1. *: p <0.05, **: p <0.01 by Dunnett’s multiple t -test.

# al . reported the dysfunction of feedback inhibition in the rat hippocampus following exposure to 1BP 11, 22, 23) 

# . It is thought that paired-pulse inhibition of the postsynaptic membrane is caused by decrease of GABA release due to low storage levels of GABA. The finding of Fueta et al

. is compatible with our results, because GABA was decreased in the hippocampus in both Cases 1 and 2 of our 1BP exposure.

In both Cases 1 and 2, aspartate and glutamine

increased and taurine decreased in almost all brain regions. Glutamine is not a neurotransmitter, but it is a precursor and a metabolite of glutamate, an excitatory neurotransmitter. In our previous studies 24–27) 

# , the inhalation exposure of rats to toluene, xylene, trichloroethylene, tetrachloroethylene, and methyl bromide increased brain glutamine. Exposure to many kinds of chemicals seems to increase brain glutamine. Glutamine is synthesized from ammonia and glutamate by glutamine synthetase.Because ammonia is toxic to the nervous system, glutamine may have increased as a result of detoxification in the brain after chemical exposure. Aspartate is known as one of excitatory neurotransmitters 28, 29) 

# , an increase in locomotor activity was observed in rats exposed to

1BP 12) 

# . Such behavioral changes may be related to the increase in cerebral aspartate observed in this study. The level of glutamate, another excitatory amino acid was not affected by 1BP in almost all brain regions in Case 2. The midbrain content of glutamate was reduced significantly at 50 and 200 ppm 1BP exposure by the Dunnett’s multiple t -test, however, the changes occurred only in one brain region and were not dose-dependent,and no statistical significance in these changes were indicated by the Kruskal-Wallis test. This suggests that the changes in glutamate may not be significant. 1-BROMOPROPANE AND BRAIN NEUROTRANSMITTERS IN MALE RATS 

# 353

Fig. 3. Effects of 1BP exposure on brain levels of amino acids in male rats at 2 h after the end of 3 wk inhalation exposure. The amounts of amino acids are expressed in μmoles/g tissue. Results are shown as means ± SD (n=5, except the control group for hippocampal GABA, n=4.). **: p <0.01 by Dunnett’s multiple t -test.

# 354 M SUDA et al

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Industrial Health 2008, 46 , 348–359Fig. 4. Effects of 1BP exposure on brain levels of ACh, DA, DOPAC and HVA, and DOPAC/DA and HVA/DA ratios in male rats at 19 h after the end of 3 wk inhalation exposure.See legend for Fig. 1. Results are shown as means ± SD (n=3–5). *:p<0.05 by Dunnett’s multiplet-test.

# 1-BROMOPROPANE AND BRAIN NEUROTRANSMITTERS IN MALE RATS 

# 355Fig. 5. Effects of 1BP exposure on brain levels of NE, MHPG, 5HT, and 5HIAA, and MHPG/NE and 5HIAA/5HT ratios in male rats at 19h after the end of 3 wk inhalation exposure.See legend for Fig. 1. Results are shown as means ± SD (n=3–5). *:p<0.05, **:p<0.01 by Dunnett’s multiplet-test.

# 356 M SUDA et al

.

Industrial Health 2008, 46 , 348–359Fig. 6. Effects of 1BP exposure on brain levels of amino acids in male rats at 19 h after the end of 3 wk inhalation exposure.See legend for Fig. 3. Results are shown as means ± SD (n=5). *:p<0.05, **:p<0.01 by Dunnett’s multiplet-test.

# 1-BROMOPROPANE AND BRAIN NEUROTRANSMITTERS IN MALE RATS 

# 357Table 1. Effects of 1BP exposure on the brain contents of major free amino acids in male rats at 19 h after the end of 3 wk inhalation exposureTyrosineMethionineCystathionineSerineThreonineAlanine β-AlanineFrontalcortexControl0.0900±0.01160.0276±0.00270.0260±0.00161.028±0.0390.428±0.0240.499±0.0460.0577±0.002950ppm0.0940±0.01090.0267±0.00100.0273±0.00331.054±0.0530.421±0.0060.506±0.0280.0583±0.0023200ppm0.0979±0.01110.0343±0.00970.0263±0.00221.042±0.0430.417±0.0150.591±0.1630.0561±0.00401,000ppm0.0903±0.01150.0304±0.00740.0416±0.00501.178±0.066**0.606±0.012**0.457±0.0440.0797±0.0077**OccipitalcortexControl0.0788±0.00490.0375±0.00300.0405±0.00381.010±0.0370.390±0.0080.509±0.0890.0501±0.002950ppm0.0776±0.01050.0313±0.00350.0386±0.00080.989±0.0470.368±0.0330.475±0.0540.0509±0.0041200ppm0.0853±0.02060.0342±0.01250.0357±0.0015*0.997±0.0250.376±0.0400.559±0.1870.0516±0.00801,000ppm0.0739±0.00890.0350±0.00430.0512±0.0017**1.170±0.055**0.519±0.023**0.451±0.0070.0692±0.0100**HippocampusControl0.0735±0.00840.0260±0.00580.0352±0.00570.948±0.0220.394±0.0170.581±0.0940.0442±0.002450ppm0.0761±0.01030.0227±0.00290.0294±0.00340.966±0.0290.400±0.0200.581±0.0630.0439±0.0011200ppm0.0661±0.01030.0246±0.00670.0287±0.00540.882±0.1380.360±0.0370.561±0.0330.0400±0.00731,000ppm0.0687±0.01080.0230±0.00450.0392±0.00261.031±0.0540.509±0.025**0.540±0.0460.0601±0.0070**StriatumControl0.0866±0.01130.0401±0.00440.0636±0.00641.006±0.0430.475±0.0250.481±0.0570.0664±0.004050ppm0.0983±0.01540.0338±0.00210.0566±0.00651.009±0.0250.467±0.0270.462±0.0620.0693±0.0063200ppm0.0921±0.01840.0437±0.00840.0667±0.00691.010±0.0660.460±0.0340.513±0.1730.0649±0.00931,000ppm0.0872±0.01280.0338±0.00600.0756±0.01231.083±0.1030.591±0.071**0.391±0.0610.1024±0.0175**MidbrainControl0.0827±0.01140.0366±0.00440.0653±0.00960.577±0.0890.428±0.0350.355±0.1130.0843±0.009250ppm0.0856±0.01330.0341±0.00870.0621±0.00620.458±0.054*0.361±0.052*0.310±0.1150.0775±0.0058200ppm0.0861±0.02270.0341±0.01450.0612±0.00470.467±0.021*0.350±0.030*0.350±0.2180.0803±0.00691,000ppm0.0703±0.00530.0266±0.00340.0788±0.0071*0.647±0.0660.486±0.0420.263±0.0410.1240±0.0130**HypothalamusControl0.0746±0.01180.0402±0.00680.0788±0.02610.492±0.0220.367±0.0290.342±0.0500.0887±0.004850ppm0.0757±0.00580.0276±0.00280.0473±0.0056*0.464±0.0260.338±0.0360.299±0.0570.0832±0.0058200ppm0.0866±0.02500.0428±0.01480.0714±0.02090.508±0.0540.338±0.0380.417±0.2050.0857±0.00661,000ppm0.0704±0.00960.0253±0.0016*0.0588±0.00800.628±0.047**0.430±0.042*0.274±0.0380.1294±0.0135**Medulla-oblongataControl0.0759±0.02390.0343±0.00950.1171±0.03100.350±0.0970.380±0.1120.326±0.1320.0463±0.009750ppm0.0658±0.01060.0292±0.00380.1018±0.01700.296±0.0360.315±0.0350.277±0.0510.0406±0.0067200ppm0.0786±0.01970.0336±0.01250.1083±0.01110.344±0.0740.356±0.0820.335±0.1750.0508±0.00691,000ppm0.0629±0.00580.0290±0.00400.1359±0.01390.457±0.0590.440±0.0460.237±0.0280.0652±0.0090**CerebellumControl0.0719±0.00710.0260±0.00810.3355±0.02250.488±0.0240.369±0.0260.380±0.1380.0326±0.001550ppm0.0733±0.00830.0218±0.00510.3307±0.02670.475±0.0230.349±0.0210.371±0.1230.0327±0.0009200ppm0.0727±0.01860.0245±0.00630.3145±0.03400.476±0.0220.340±0.0290.398±0.2280.0329±0.00121,000ppm0.0585±0.00620.0226±0.00310.4334±0.0291**0.694±0.042**0.464±0.018**0.248±0.0080.0461±0.0056**The amounts of amino acids are expressed in μmoles/g tissue. Results are shown as means ± SD (n=5). *:p<0.05, **:p<0.01 by Dunnett’s multiplet-test.

# The brain level of taurine was lowered significantly in almost all brain regions at 1,000 ppm 1BP exposure inCase 2. The decrease in taurine was observed in the hippocampus and midbrain at 200 ppm and lower concentrations of 1BP exposure. According to Wang et al ., creatine kinase activity was decreased dose-dependently by exposure to 1BP at 200 ppm or higher 30) 

# . As an index for detecting the influence of 1BP on the nervous system, the level of taurine is understood to be almost equal toor higher than that of creatine kinase activity in sensitivity. Among free amino acids in the rat brain, taurine belongs to the group in which the brain content levels are very high. Taurine is involved in many functions suchas detoxification, cholesterol metabolism, and neuromodulation 31) 

# . It is very likely that taurine is a large source

for transamination 32) 

# . At present, it is difficult to state which function of taurine was affected by 1BP exposure. As shown in Table 1, significant changes were observedin some amino acids, particularly at 1,000 ppm 1BP exposure. Almost all the amino acids in Table 1 are involved

in amino acid transamination 33–35) 

# . Widespread changes of these amino acids induced by 1BP exposure may have led to the changes in transamination in the brain. Alternatively, the changes in transamination may have produced the changes in amino acids contents. In contrast to biogenic amines like DA, 5HT, etc., the metabolism of amino acids is complex, and amino acid contents are considered to be constant. Contrary to ourexpectation, this study elucidated that the amino acid contents of the rat brain are changeable, but it is not clearwhether the widespread changes in amino acids are specific to 1BP exposure. It seems at least that the increase in glutamine in the brain is a common effect of exposureto many chemicals. Further studies are necessary to elucidate the mechanism of 1BP influence on brain substances.



# Acknowledgements

We appreciate the considerable contribution of Yusuke Tsujimura, Jun Sato, and Junko Kuwahara. The present study was conducted as a part of a researchproject at JNIOSH on “Development of biological indicators and health management under circumstances of low exposure level of hazardous factors” (P17-02).



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