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Changes in free amino acid contents of rat brain induced by exposure to methyl bromide
Letters, 15 (1983) 317-321
Toxicology Elsevier
Biomedical
317
Press
CHANGES IN FREE AMINO ACID CONTENTS OF RAT BRAIN INDUCED BY EXPOSURE TO METHYL BROMIDE (Glutamine; aspartic acid; alanine; glycine)
TAKESHI
HONMA,
AYAKO
SUDO, MUNEYUKI
MIYAGAWA,
MITSUO
SAT0
and HIROMICHI
HASEGAWA National Institute of Industrial Health, 21-1, Nagao Gchome, (Received
June
(Revision
received
14th,
(Accepted
October
Tama-ku, Kawasaki 213 (Japan)
1982)
September lst,
27th,
1982)
1982)
SUMMARY Rats were exposed Changes
to methyl
in free amino
chromatography and long-term
acid
bromide contents
(MB) for 24 h at lo-120 of rat
midbrain
were
ppm or for 3 weeks at I-IO
measured
by high-performance
ppm. liquid
(HPLC). MB increased glutamine and aspartic acid contents dose-dependently by shortexposure. Alanine content was markedly increased by long-term exposure to IO ppm MB.
Glycine
was dose-dependently
nervous
system is discussed
increased, in relation
except at 120 ppm. The harmful to the changes
in amino
effect of MB on the central
acid metabolism.
INTRODUCTION
MB (CHJBr), a compound used as a fumigant and methylating agent, may cause severe CNS disorders in man, e.g. ataxia, disturbance of consciousness, convulsion, delusion and hallucination, following inhalation [l-3]. In rat studies (unpublished observation), MB disturbed locomotor activity and in other studies has been shown to induce conditioned taste aversion [4]. We have already reported that the exposure of rats to MB produces a reduction of NE content of the brain [5]. NE is known to play a role as a neurotransmitter in the CNS. These results show that MB may affect brain function in both man and animals.
Abbreviations: CNS, central nervous system; GABA, y-aminobutyric liquid chromatograph(y); MB, methyl bromide; NE, norepinephrine;
0378-4274/83/OOOC-0000/$03.00
0 Elsevier
Biomedical
Press
acid; HPLC, high-performance TLV, threshold limit value.
318
Tri- and tetrachloroethylene, toluene and hexane, which may affect the CNS, produce significant changes in free amino acid contents of rat brain [6, 71. These results suggest the possibility that many substances, toxic to the CNS may act through a disturbance of amino acid metabolism. There is much evidence to support the hypothesis that some amino acids such as GABA, glutamic acid or glycine may play a role as a neurotransmitter in the CNS. In the present report, we have described the changes in amino acid contents of the brain induced by exposure to MB and these observations are discussed in relation to MB, CNS toxicity. MATERIALS
AND METHODS
Groups of rats (5 to 6 male rats/group: Charles River Japan, SD strain), 250-300 g at 8-12 weeks of age were barrier-maintained in inhalation chambers under conditions of 12 h light and 12 h dark at 24” + 1°C and 55% -e 5% humidity. Food and water were provided ad lib. Rats were exposed to MB gas in 60-l stainless steel chambers. The concentration of MB, which was adjusted to the desired concentration by mixing with appropriate volume of air, was monitored using an FID gas chromatograph equipped with an automatic sampling device. To prevent post mortem change of neurotransmitter substances by inactivating the metabolizing enzymes, the rat head was irradiated by microwave and the brain temperature raised. Immediately after the exposure, the rat brain was irradiated by microwave power (5.0 kW, 1.9 s) in a microwave applicator (Muromachi Kikai Co., Tokyo, Japan). The midbrain was dissected on an ice-cold plate by the method of Glowinski and Iversen [8], and stored at - 80°C. Detailed assay methods for amino acids have been described previously [6]. The midbrain tissue was homogenized in 4 ml of 0.1 N HC104 containing 20 ,uI of 50 mM phosphoserine as an internal standard. The homogenate was centrifuged, and 20 ~1 of the supernatant was applied to an HPLC system (Shimazu Corp., Kyoto, Japan) equipped with an automatic sampling device. Amino acids were eluted with citrate buffer and the eluate was allowed to react with o-phthalaldehyde dissolved in borate buffer. The amino acid concentration was estimated from fluorescence intensity using a fluorescence detector. The results were analysed statistically using Student’s t-test. RESULTS
Groups of rats were exposed to air, 10, 20, 40, 60, 100 and 120 ppm of MB, respectively, for 24 h and post mortem examinations carried out immediately after exposure. An additional group of rats was exposed to 120 ppm of MB for 24 h and held for 24 h prior to post mortem examinations. A further 4 groups of rats were
319
Gln
Fig. 1, The changes in amino acid contents of rat midbrain induced by methyl bromide exposure. Mean k S.E.M. values of amino acid contents of exposed groups were expressed as percentages of mean amino acid contents of control group. The columns and vertical bars represent percent changes of means from control (IO@%) value and S.E.M. values, respectively. Hatched columns mean significant difference from control value by f-test. (Left, second diagram from top: Glu.)
exposed to air, 1, 5 and 10 ppm of MB, respectively, for 3 weeks and killed immediately after exposure. Fig. 1 shows the changes in amino acid contents of rat midbrain induced by MB exposure. Mean amino acid content (per g tissue) of control group was arbitrarily set to 100% and percentage changes in mean amino acid contents of MB-exposed groups were calculated. Glutamine and aspartic acid contents of rat brain increased dose-dependently following short- and Iong-term exposure to MB. Alanine content was markedly increased following long-term exposure to 10 ppm MB. Glycine was increased by short-term exposure to MB at 40,60 and 100 ppm, but was not changed by 120 ppm MB. Changes in GABA and taurine contents did not follow a consistent trend after either short- or long-term exposure. Glutamic acid content of the brain was unaltered by MB. Mean amino acid contents of control groups ranged from 2.53-2.99 pmolfg for GABA, 8.45-8.57 pmollg for glutamic acid, 4.53-6.27 FmoIlg for glutamine,
320
2.84-3.53 pmol/g for taurine, 2.38-2.75 kmol/g for aspartic acid, 1.24-1.86 pmol/g for glycine, 0.206-0.298 hmol/g for alanine. DISCUSSION
Glutamine content of rat brain was increased following short- or long-term exposure to tri- and tetrachloroethylene, toluene and hexane [6, 71. In the present study, MB increased the levels of glutamine in the brain. These results suggest that the levels of glutamine in the brain may be increased by exposure to a range of neurotoxic substances. MB exposure increased alanine, especially following longterm (3 weeks) exposure. It has been demonstrated that MB is capable of methylating enzymes containing sulfhydryl (SH) groups, e.g. succinate dehydrogenase [9]. This could result in the inhibition of TCA cycle activity leading to an elevation of alanine levels [lo]. The results of our studies would suggest that MB exposure may increase the tissue levels of alanine through TCA cycle inhibition. Similarly, aspartic acid levels may be increased through an increased availability of oxaloacetic acid, a member of the TCA cycle, under these conditions. An increase in glutamine synthesis may arise from elevated levels of ammonia, the latter being increased by MB exposure. If indeed ammonia levels were increased in the brain [ 111, it is likely that glycine metabolism is inhibited, resulting in elevated levels of this metabolite. Increase in glutamine and aspartic acid induced by short-term (24 h) exposure to 120 ppm MB lasted for at least 24 h following the cessation of exposure. These results indicate that the effect of MB exposure on amino acid contents is long-lasting and analogous to the changes observed in monoamine levels [5]. Aspartic acid was significantly increased at 20 ppm by short-term exposure of MB. Following long-term exposure to MB, significant increases in aspartic acid and alanine contents were observed at 5 ppm. The TLV for MB has been set at 15 ppm by the American Conference of Governmental Industrial Hygienists. The results of the present experiment have indicated that the exposure to MB may produce toxic effects on the CNS at a lower concentration than 15 ppm. ACKNOWLEDGEMENTS
The authors thank Miss M. Suda for her technical assistance.
REFERENCES
1 W.O.
Negherbon,
Methyl
bromide
(bromomethane;
monobromomethane),
Toxicology, Vol. III, Saunders, Philadelphia, 1959, pp. 477-484. 2 W.F. von Oettingen, Halogenated Hydrocarbons ‘Methyl Bromide’, 3 D. Hunter,
Methyl Bromide,
The Diseases of Occupations,
English
Elsevier, University
in Handbook Amsterdam, Press, London,
of
1965. 1975.
321 4 M. Miyagawa, Conditioned taste aversion induced by inhalation exposure to methyl bromide in rats, Toxicol. Lett., 10 (1982) 411-416. 5 T. Honma, A. Sudo, M. Miyagawa, M. Sato and H. Hasegawa, Significant changes in monoamines in rat brain induced by exposure to methyl bromide, Neurobehav. Toxicol. Teratol., 4 (1982) in press. 6 T. Honma, H. Hasegawa, M. Sato and A. Sudo, Changes of free amino acid content in rat brain after exposure to trichloroethylene and tetrachloroethylene, Indust. Health, 18 (1980) l-7. 7 T. Honma, M. Miyagawa, M. Sato and H. Hasegawa, Increase in glutamine content of rat midbrain induced by short-term exposure to toluene and hexane, Indust. Health, 20 (1982) 109115. 8 J. Glowinski and L.L. Iversen, Regional studies of catecholamines in the rat brain, I. The disposition of [‘Hlnorepinephrine, [‘Hldopamine and [‘HIDOPA in various regions of the brain, J. Neurochem., 13 (1966) 655-669. 9 S.E. Lewis, Inhibition of SH enzymes by methyl bromide, Nature, 161 (1948) 692-693. 10 R. Balrlzs, Y. Machiyama and A.J. Patel, Compartmentation and the metabolism of y-aminobutyric acid, in R. Baliicz and J.E. Cremer (Eds.), Metabolic Compartmentation in the Brain, Macmillan, London, 1973, p. 57. 11 Y. Tsukada, Amino acid metabolism and its relation to brain functions, Prop. Brain Res., 21A (1966) 268-291.
Toxicology Elsevier
Biomedical
317
Press
CHANGES IN FREE AMINO ACID CONTENTS OF RAT BRAIN INDUCED BY EXPOSURE TO METHYL BROMIDE (Glutamine; aspartic acid; alanine; glycine)
TAKESHI
HONMA,
AYAKO
SUDO, MUNEYUKI
MIYAGAWA,
MITSUO
SAT0
and HIROMICHI
HASEGAWA National Institute of Industrial Health, 21-1, Nagao Gchome, (Received
June
(Revision
received
14th,
(Accepted
October
Tama-ku, Kawasaki 213 (Japan)
1982)
September lst,
27th,
1982)
1982)
SUMMARY Rats were exposed Changes
to methyl
in free amino
chromatography and long-term
acid
bromide contents
(MB) for 24 h at lo-120 of rat
midbrain
were
ppm or for 3 weeks at I-IO
measured
by high-performance
ppm. liquid
(HPLC). MB increased glutamine and aspartic acid contents dose-dependently by shortexposure. Alanine content was markedly increased by long-term exposure to IO ppm MB.
Glycine
was dose-dependently
nervous
system is discussed
increased, in relation
except at 120 ppm. The harmful to the changes
in amino
effect of MB on the central
acid metabolism.
INTRODUCTION
MB (CHJBr), a compound used as a fumigant and methylating agent, may cause severe CNS disorders in man, e.g. ataxia, disturbance of consciousness, convulsion, delusion and hallucination, following inhalation [l-3]. In rat studies (unpublished observation), MB disturbed locomotor activity and in other studies has been shown to induce conditioned taste aversion [4]. We have already reported that the exposure of rats to MB produces a reduction of NE content of the brain [5]. NE is known to play a role as a neurotransmitter in the CNS. These results show that MB may affect brain function in both man and animals.
Abbreviations: CNS, central nervous system; GABA, y-aminobutyric liquid chromatograph(y); MB, methyl bromide; NE, norepinephrine;
0378-4274/83/OOOC-0000/$03.00
0 Elsevier
Biomedical
Press
acid; HPLC, high-performance TLV, threshold limit value.
318
Tri- and tetrachloroethylene, toluene and hexane, which may affect the CNS, produce significant changes in free amino acid contents of rat brain [6, 71. These results suggest the possibility that many substances, toxic to the CNS may act through a disturbance of amino acid metabolism. There is much evidence to support the hypothesis that some amino acids such as GABA, glutamic acid or glycine may play a role as a neurotransmitter in the CNS. In the present report, we have described the changes in amino acid contents of the brain induced by exposure to MB and these observations are discussed in relation to MB, CNS toxicity. MATERIALS
AND METHODS
Groups of rats (5 to 6 male rats/group: Charles River Japan, SD strain), 250-300 g at 8-12 weeks of age were barrier-maintained in inhalation chambers under conditions of 12 h light and 12 h dark at 24” + 1°C and 55% -e 5% humidity. Food and water were provided ad lib. Rats were exposed to MB gas in 60-l stainless steel chambers. The concentration of MB, which was adjusted to the desired concentration by mixing with appropriate volume of air, was monitored using an FID gas chromatograph equipped with an automatic sampling device. To prevent post mortem change of neurotransmitter substances by inactivating the metabolizing enzymes, the rat head was irradiated by microwave and the brain temperature raised. Immediately after the exposure, the rat brain was irradiated by microwave power (5.0 kW, 1.9 s) in a microwave applicator (Muromachi Kikai Co., Tokyo, Japan). The midbrain was dissected on an ice-cold plate by the method of Glowinski and Iversen [8], and stored at - 80°C. Detailed assay methods for amino acids have been described previously [6]. The midbrain tissue was homogenized in 4 ml of 0.1 N HC104 containing 20 ,uI of 50 mM phosphoserine as an internal standard. The homogenate was centrifuged, and 20 ~1 of the supernatant was applied to an HPLC system (Shimazu Corp., Kyoto, Japan) equipped with an automatic sampling device. Amino acids were eluted with citrate buffer and the eluate was allowed to react with o-phthalaldehyde dissolved in borate buffer. The amino acid concentration was estimated from fluorescence intensity using a fluorescence detector. The results were analysed statistically using Student’s t-test. RESULTS
Groups of rats were exposed to air, 10, 20, 40, 60, 100 and 120 ppm of MB, respectively, for 24 h and post mortem examinations carried out immediately after exposure. An additional group of rats was exposed to 120 ppm of MB for 24 h and held for 24 h prior to post mortem examinations. A further 4 groups of rats were
319
Gln
Fig. 1, The changes in amino acid contents of rat midbrain induced by methyl bromide exposure. Mean k S.E.M. values of amino acid contents of exposed groups were expressed as percentages of mean amino acid contents of control group. The columns and vertical bars represent percent changes of means from control (IO@%) value and S.E.M. values, respectively. Hatched columns mean significant difference from control value by f-test. (Left, second diagram from top: Glu.)
exposed to air, 1, 5 and 10 ppm of MB, respectively, for 3 weeks and killed immediately after exposure. Fig. 1 shows the changes in amino acid contents of rat midbrain induced by MB exposure. Mean amino acid content (per g tissue) of control group was arbitrarily set to 100% and percentage changes in mean amino acid contents of MB-exposed groups were calculated. Glutamine and aspartic acid contents of rat brain increased dose-dependently following short- and Iong-term exposure to MB. Alanine content was markedly increased following long-term exposure to 10 ppm MB. Glycine was increased by short-term exposure to MB at 40,60 and 100 ppm, but was not changed by 120 ppm MB. Changes in GABA and taurine contents did not follow a consistent trend after either short- or long-term exposure. Glutamic acid content of the brain was unaltered by MB. Mean amino acid contents of control groups ranged from 2.53-2.99 pmolfg for GABA, 8.45-8.57 pmollg for glutamic acid, 4.53-6.27 FmoIlg for glutamine,
320
2.84-3.53 pmol/g for taurine, 2.38-2.75 kmol/g for aspartic acid, 1.24-1.86 pmol/g for glycine, 0.206-0.298 hmol/g for alanine. DISCUSSION
Glutamine content of rat brain was increased following short- or long-term exposure to tri- and tetrachloroethylene, toluene and hexane [6, 71. In the present study, MB increased the levels of glutamine in the brain. These results suggest that the levels of glutamine in the brain may be increased by exposure to a range of neurotoxic substances. MB exposure increased alanine, especially following longterm (3 weeks) exposure. It has been demonstrated that MB is capable of methylating enzymes containing sulfhydryl (SH) groups, e.g. succinate dehydrogenase [9]. This could result in the inhibition of TCA cycle activity leading to an elevation of alanine levels [lo]. The results of our studies would suggest that MB exposure may increase the tissue levels of alanine through TCA cycle inhibition. Similarly, aspartic acid levels may be increased through an increased availability of oxaloacetic acid, a member of the TCA cycle, under these conditions. An increase in glutamine synthesis may arise from elevated levels of ammonia, the latter being increased by MB exposure. If indeed ammonia levels were increased in the brain [ 111, it is likely that glycine metabolism is inhibited, resulting in elevated levels of this metabolite. Increase in glutamine and aspartic acid induced by short-term (24 h) exposure to 120 ppm MB lasted for at least 24 h following the cessation of exposure. These results indicate that the effect of MB exposure on amino acid contents is long-lasting and analogous to the changes observed in monoamine levels [5]. Aspartic acid was significantly increased at 20 ppm by short-term exposure of MB. Following long-term exposure to MB, significant increases in aspartic acid and alanine contents were observed at 5 ppm. The TLV for MB has been set at 15 ppm by the American Conference of Governmental Industrial Hygienists. The results of the present experiment have indicated that the exposure to MB may produce toxic effects on the CNS at a lower concentration than 15 ppm. ACKNOWLEDGEMENTS
The authors thank Miss M. Suda for her technical assistance.
REFERENCES
1 W.O.
Negherbon,
Methyl
bromide
(bromomethane;
monobromomethane),
Toxicology, Vol. III, Saunders, Philadelphia, 1959, pp. 477-484. 2 W.F. von Oettingen, Halogenated Hydrocarbons ‘Methyl Bromide’, 3 D. Hunter,
Methyl Bromide,
The Diseases of Occupations,
English
Elsevier, University
in Handbook Amsterdam, Press, London,
of
1965. 1975.
321 4 M. Miyagawa, Conditioned taste aversion induced by inhalation exposure to methyl bromide in rats, Toxicol. Lett., 10 (1982) 411-416. 5 T. Honma, A. Sudo, M. Miyagawa, M. Sato and H. Hasegawa, Significant changes in monoamines in rat brain induced by exposure to methyl bromide, Neurobehav. Toxicol. Teratol., 4 (1982) in press. 6 T. Honma, H. Hasegawa, M. Sato and A. Sudo, Changes of free amino acid content in rat brain after exposure to trichloroethylene and tetrachloroethylene, Indust. Health, 18 (1980) l-7. 7 T. Honma, M. Miyagawa, M. Sato and H. Hasegawa, Increase in glutamine content of rat midbrain induced by short-term exposure to toluene and hexane, Indust. Health, 20 (1982) 109115. 8 J. Glowinski and L.L. Iversen, Regional studies of catecholamines in the rat brain, I. The disposition of [‘Hlnorepinephrine, [‘Hldopamine and [‘HIDOPA in various regions of the brain, J. Neurochem., 13 (1966) 655-669. 9 S.E. Lewis, Inhibition of SH enzymes by methyl bromide, Nature, 161 (1948) 692-693. 10 R. Balrlzs, Y. Machiyama and A.J. Patel, Compartmentation and the metabolism of y-aminobutyric acid, in R. Baliicz and J.E. Cremer (Eds.), Metabolic Compartmentation in the Brain, Macmillan, London, 1973, p. 57. 11 Y. Tsukada, Amino acid metabolism and its relation to brain functions, Prop. Brain Res., 21A (1966) 268-291.