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Glutamic acid and sodium levels in the nucleus arcuatus of the hypothalamus of adult and infant guinea pigs after oral monosodium glutamate
313
Toxicology Letters, 4 (1979) 313-316 o E~ev~er~N~rth-~oiland Biomedical Press
GLUTAMIC ACID AND SODOM LEVELS IN THE NUCLEUS ARCUATUS OF THE HYPOTHALAMUS OF ADULT AND INFANT GUINEA PIGS AFTER ORAL MONOSODIUM GLUTAMATE
L. AIRGLDI and S. GARATTINI* kstituta
di Ricer&e
~~r~~cul~gi~~e
‘Murio Negri’, Via Eritrea, 6.2, 2~~57”~j~a~
f’ftaly,l
(Received June 28th, 1979) (Accepted July lst, 1979)
_____-____
_ -_--
SUMMARY
Monosodium glutamate (MSG) administered orally at 4 g/kg body weight (b.wt.) as a 20% solution (w/v) to 55-66-day-old guinea pigs and at 2 g/kg b.wt. as a 20% solution (w/v) to 3-day-old guinea pigs did not increase glutamir: acid (GA) or sodium levels in the nucleus arcuatus of the hypothalamus (NAN) or in the thalamus lateralis (TL). Sodium chloride given by gavage at doses of sodium equimolar to MSG resulted in an increase of sodium content only in the NAH of infant guinea pigs. ~_..
In a recent paper fl] we reported that GA did not accumulate in the NAH of adult or infant rats after oral administration of MSG. These findings are at variance with those obtained by Perez et al. in mice [Z] and suggest that in rats the neurotoxic effect of MSG cannot be explained on the basis of an increase of GA in NAH. Our primary interest was to assess whether or not other laboratory animals susceptible to MSG neurotoxi~i~, e.g. guinea pigs f3, 4] accumulate GA in NAH. The present paper reports the effect of orally administered MSG on plasma and NAH levels of GA and sodium in infant and adult guinea pigs. The effect of oral NaCl on plasma and NAH sodium content is also reported. MATERIALS
AND METHODS
Male albino-English guinea pigs 55 to 60 days old and albino-English guinea pigs of both sexes, 3 days old, were used (Pellizzari Breeding, Italy), Animals *To whom correspondence is to be sent. Abbreviations: GA, glutamic acid; MSG, monosodium glutamate; NAH, nucleus arcuatus of the hypothalamus; TL, thalamus lateralis.
314
were housed in standard conditions (temperature 20 f l”C, relative humidity 60%, 12 h light/dark cycles) and had unrestricted access to food and water until the experiment was started. Adult guinea pigs were given MSG by gavage at 4 g/kg b.wt. as a 20% solution (w/v); a second group of adult animals was given NaCl by gavage at 1.37 g/kg b.wt.; a dose equimolar for sodium to 4 g/kg b.wt. of MSG. The concentration of sodium in the NaCl solution was the same as in the MSG solution. Control animals received tap water. 3-day-old guinea pigs were housed 4 per cage with a lactating mother until the experiment started. MSG was administered by gavage at 2 g/kg b.wt. as a 20% solution (w/v). NaCl was administered by gavage at 0.68 g/kg b.wt. which is equimolar for sodium to 2 g/kg b.wt. of MSG. Sodium concentration in the NaCl solution was the same as in the 20% MSG solution. Control animals received tap water. Adult and infant animals were killed by decapitation 60 and 180 min after treatment. Blood samples were collected from the cervical wound in heparinized tubes and brains removed, immediately frozen in powdered dry ice and mounted on the specimen holder of a cryostat. Frontal sections 300 ,um thick were cut at - 10°C. The correct localization of NAH was ascertained [5]. NAH was isolated by the punching technique described by Palkovits [6] using a 400 pm needle. The TL was dissected as a control area. Tissue preparation was as previously described [l]. GA was determined in plasma, NAH and TL by the method of Young and Lowry [7]. Plasma, NAH and TL sodium levels were measured with a Perkin-Elmer Atomic Absorption Spectrophotometer model 603. NAH and TL protein levels were determined by Bradford’s method IS]. RESULTS
GA levels in plasma, NAH and TL of 60- and 3-day-old guinea pigs after oral MSG are reported in Tables I and II respectively. In spite of the marked inTABLE I BO-DAY-OLD GUINEA PIGS GA in plasma, nucleus arcuatus and thalamus lateralis after MSG (4 gfkg by gavage) Min after treatment
Number of animals
Plasma nmol/ml + S.E.
Nucleus arcuatus nmol/mg protein f S.E.
Thaiamus lateralis nmol/mg protein + S.E.
0 60 180
4 4 4
251+ 8 1452 + 635a 383 f 182
253 zi 28 192 f 30 206 * 24
265 f 26 230 f 40 278 i 11
aP < 0.06compared to control values (Duncan’s test).
315 TABLE II 3-DAY-OLD GUINEA PIGS GA in plasma, nucleus arcuatus and thalamus lateralis after MSG (2 g/kg by gavage) ._.-...-.--I___-.Min after treatment
animals
Plasma nmol/ml * S.E.
Nucleus arcuatus nmol/mg protein I S.E.
Thalamus lateralis nmol/ml protein * S.E.
4 4 4
328 * 51 7912 i 27Ha 252 2 34
212 rf.15 215 ?r 21 21’7 r 19
179 + 7 209 + 33 198 t 6
Number Of
0
60 180
‘J’ < 0.01 compared to control values {Duncan’s test). TABLE
IKI
GUDAY-OLD
GUKNEA PIGS
Sodium in plasma, nucleus arcuatus and thalamus later&is after MSG or NaCl P.O. MiO after treatment
Number of animaIs
0
4
Nucleus arouatus nEq./mg protein f SE.
106 + 3
776 t 120
MSG -_
~60 180
TABLE
Plasma &Eg. /ml f S.E.
4 4
108 + 11 121 i 3
Thalamus lateralis nEq./mg protein t S.E. -------569 * 45
NaCl ..~..
MSG
NaCK -
MSG I__-..
116 r 3 115+ 2
863 t 104 824 f: 55
693 i 144 x119 i 144
595 + 62 678 * 71
NaCI .-~ 758 f 53 551 k 42
IV
3-DAY-OLD
GUINEA
PIGS
Sodium in pIanna, nucleus arcuatus and thabunus laterahs after MSG or NaCl p.o. ~..__l___ _~_.._ Mm after -treatment
Number of animals
0
4
f S.E.
111 t 2
---~ 60 180
Plasma fig&/ml
4 4
Nucleus arcuatus nEq,/mg protein t S.E.
Thalamus lateralis nEq./mg protein f S.E.
974 t 183
.__._.____._~ 703 t 28
MSG
N&l
MSG
NaCK
MSG
NaCI
134 f 11 123* 3
123 + 4 125 t 4
969 k 100 1098 i 132
759 t 67 1573 i 207a
791 i 78 868 + 148
830 f 135 972 + 220
a P < 0.05 compared to control value (Duncan’s test).
crease of plasma GA in both age-groups, 60 min after MSG administration, GA did not accumulate in the NAH or TL of either adult or infant guinea Tables III and IV report sodium concentration in plasma, NAH and TL MSG or NaCl given by gavage to adult and infant guinea pigs respectively. change in sodium levels was observed in adult guinea pigs, but in S-day-old animals 180 min after NaCl administration, sodium increased in the NAH, MSG had no effect on plasma, NAH or TL sodium concentrations.
pigs. after No Qral
316
No GA accumulation was observed in the NM of either adult or infant guinea pigs after oral admin~t~tion of MSG at doses reportedly neurotaxic for 3-day-old animals 13, 41. The present results parallel those previously reported on adult and infant rats [1] and suggest that it may not be necessary for GA levels to be increased in discrete brain areas for neuronal lesions to occur. The mechanism of neurotoxicity suggested for mice cannot be generalized. In this study, as in fl]_ we cannot ascribe the neurotoxic effect of MSG to its sodium content, since no change was observed in sodium levels in NAH and TL of either adult or infant guinea pigs after oral MSG.
The authors wish to thank Dr. A. ~av~la~o and Mr. M. Fittipalcti (Chemical Department of the Laboratorio Provinciale di Igiene e Profilassi, Milan, Italy} for kindly providing the facilities and technical assistance for sodium determinations. MSG was kindly supplied by the Ajinomoto Company. REFERENCES 1 L, Airofdi, M. Salmona, P. Ghe22i and S. Garattini, Ghztamic acid and sodium IevcIs in the nucleus arcuatus of the hypoth~~u& of adult and infant rats after oral monosodium glutamate, Toxicol. Lett., 3 (1979) 121--126. 2 V.J. Perez and J.W. Gfney, A~~urnu~at~onof glutamic acid in the arcuate nucleus of the hypothalamus of the infant mouse following subcutaneous ad~n~tratj~n of mo~~od~urn gfutamate, J. Neurochem., 19 (1972) 3777--1782, 3 J.W. Ulney, Q.L. Ho, V. Rhee and T. DeGubareff, Neurotoxic effects of ~~~t~~~t~, N. En& 3, Med,, 289 (1973) 1374-1376. 4 R. Heywood and A.N. Worden, Glutamate toxicity in laboratory animals, in L.J. Filer Jr., S. Garattini, MR. Kare, W.A. Reynolds and R.J. Wurtman (Eds.), Glutamic Acid: Advances in Biochemistry and Physiology, Raven Press, New York, 1979, pp. 263-215. 5 T.J. Luparello, Stereotaxic Atlas of the Forebrain of the Guinea Pig, Karger, Baael, 1967. 6 M. Palkovits, Isolated removal of hypothallunic or other nuclei of the rat, Brain Ras., 59 (1973) 44Q-466. 7 R.L. Young and U.H. Lowry, Quantitative methods for measuring the histocbemical distribution of a&nine, glutamate and glutamine in brain, J. Neurocheti., 13 (1966) 786-793. 8 M.M Bradford, A rapid and sensitive method far the quantjtat~on of rn~~o~ q~~~tit~a~ of protein utilizing the prineipfe of protein-dye binding, And. 3iochem., 72 (1976) 248-254.
Toxicology Letters, 4 (1979) 313-316 o E~ev~er~N~rth-~oiland Biomedical Press
GLUTAMIC ACID AND SODOM LEVELS IN THE NUCLEUS ARCUATUS OF THE HYPOTHALAMUS OF ADULT AND INFANT GUINEA PIGS AFTER ORAL MONOSODIUM GLUTAMATE
L. AIRGLDI and S. GARATTINI* kstituta
di Ricer&e
~~r~~cul~gi~~e
‘Murio Negri’, Via Eritrea, 6.2, 2~~57”~j~a~
f’ftaly,l
(Received June 28th, 1979) (Accepted July lst, 1979)
_____-____
_ -_--
SUMMARY
Monosodium glutamate (MSG) administered orally at 4 g/kg body weight (b.wt.) as a 20% solution (w/v) to 55-66-day-old guinea pigs and at 2 g/kg b.wt. as a 20% solution (w/v) to 3-day-old guinea pigs did not increase glutamir: acid (GA) or sodium levels in the nucleus arcuatus of the hypothalamus (NAN) or in the thalamus lateralis (TL). Sodium chloride given by gavage at doses of sodium equimolar to MSG resulted in an increase of sodium content only in the NAH of infant guinea pigs. ~_..
In a recent paper fl] we reported that GA did not accumulate in the NAH of adult or infant rats after oral administration of MSG. These findings are at variance with those obtained by Perez et al. in mice [Z] and suggest that in rats the neurotoxic effect of MSG cannot be explained on the basis of an increase of GA in NAH. Our primary interest was to assess whether or not other laboratory animals susceptible to MSG neurotoxi~i~, e.g. guinea pigs f3, 4] accumulate GA in NAH. The present paper reports the effect of orally administered MSG on plasma and NAH levels of GA and sodium in infant and adult guinea pigs. The effect of oral NaCl on plasma and NAH sodium content is also reported. MATERIALS
AND METHODS
Male albino-English guinea pigs 55 to 60 days old and albino-English guinea pigs of both sexes, 3 days old, were used (Pellizzari Breeding, Italy), Animals *To whom correspondence is to be sent. Abbreviations: GA, glutamic acid; MSG, monosodium glutamate; NAH, nucleus arcuatus of the hypothalamus; TL, thalamus lateralis.
314
were housed in standard conditions (temperature 20 f l”C, relative humidity 60%, 12 h light/dark cycles) and had unrestricted access to food and water until the experiment was started. Adult guinea pigs were given MSG by gavage at 4 g/kg b.wt. as a 20% solution (w/v); a second group of adult animals was given NaCl by gavage at 1.37 g/kg b.wt.; a dose equimolar for sodium to 4 g/kg b.wt. of MSG. The concentration of sodium in the NaCl solution was the same as in the MSG solution. Control animals received tap water. 3-day-old guinea pigs were housed 4 per cage with a lactating mother until the experiment started. MSG was administered by gavage at 2 g/kg b.wt. as a 20% solution (w/v). NaCl was administered by gavage at 0.68 g/kg b.wt. which is equimolar for sodium to 2 g/kg b.wt. of MSG. Sodium concentration in the NaCl solution was the same as in the 20% MSG solution. Control animals received tap water. Adult and infant animals were killed by decapitation 60 and 180 min after treatment. Blood samples were collected from the cervical wound in heparinized tubes and brains removed, immediately frozen in powdered dry ice and mounted on the specimen holder of a cryostat. Frontal sections 300 ,um thick were cut at - 10°C. The correct localization of NAH was ascertained [5]. NAH was isolated by the punching technique described by Palkovits [6] using a 400 pm needle. The TL was dissected as a control area. Tissue preparation was as previously described [l]. GA was determined in plasma, NAH and TL by the method of Young and Lowry [7]. Plasma, NAH and TL sodium levels were measured with a Perkin-Elmer Atomic Absorption Spectrophotometer model 603. NAH and TL protein levels were determined by Bradford’s method IS]. RESULTS
GA levels in plasma, NAH and TL of 60- and 3-day-old guinea pigs after oral MSG are reported in Tables I and II respectively. In spite of the marked inTABLE I BO-DAY-OLD GUINEA PIGS GA in plasma, nucleus arcuatus and thalamus lateralis after MSG (4 gfkg by gavage) Min after treatment
Number of animals
Plasma nmol/ml + S.E.
Nucleus arcuatus nmol/mg protein f S.E.
Thaiamus lateralis nmol/mg protein + S.E.
0 60 180
4 4 4
251+ 8 1452 + 635a 383 f 182
253 zi 28 192 f 30 206 * 24
265 f 26 230 f 40 278 i 11
aP < 0.06compared to control values (Duncan’s test).
315 TABLE II 3-DAY-OLD GUINEA PIGS GA in plasma, nucleus arcuatus and thalamus lateralis after MSG (2 g/kg by gavage) ._.-...-.--I___-.Min after treatment
animals
Plasma nmol/ml * S.E.
Nucleus arcuatus nmol/mg protein I S.E.
Thalamus lateralis nmol/ml protein * S.E.
4 4 4
328 * 51 7912 i 27Ha 252 2 34
212 rf.15 215 ?r 21 21’7 r 19
179 + 7 209 + 33 198 t 6
Number Of
0
60 180
‘J’ < 0.01 compared to control values {Duncan’s test). TABLE
IKI
GUDAY-OLD
GUKNEA PIGS
Sodium in plasma, nucleus arcuatus and thalamus later&is after MSG or NaCl P.O. MiO after treatment
Number of animaIs
0
4
Nucleus arouatus nEq./mg protein f SE.
106 + 3
776 t 120
MSG -_
~60 180
TABLE
Plasma &Eg. /ml f S.E.
4 4
108 + 11 121 i 3
Thalamus lateralis nEq./mg protein t S.E. -------569 * 45
NaCl ..~..
MSG
NaCK -
MSG I__-..
116 r 3 115+ 2
863 t 104 824 f: 55
693 i 144 x119 i 144
595 + 62 678 * 71
NaCI .-~ 758 f 53 551 k 42
IV
3-DAY-OLD
GUINEA
PIGS
Sodium in pIanna, nucleus arcuatus and thabunus laterahs after MSG or NaCl p.o. ~..__l___ _~_.._ Mm after -treatment
Number of animals
0
4
f S.E.
111 t 2
---~ 60 180
Plasma fig&/ml
4 4
Nucleus arcuatus nEq,/mg protein t S.E.
Thalamus lateralis nEq./mg protein f S.E.
974 t 183
.__._.____._~ 703 t 28
MSG
N&l
MSG
NaCK
MSG
NaCI
134 f 11 123* 3
123 + 4 125 t 4
969 k 100 1098 i 132
759 t 67 1573 i 207a
791 i 78 868 + 148
830 f 135 972 + 220
a P < 0.05 compared to control value (Duncan’s test).
crease of plasma GA in both age-groups, 60 min after MSG administration, GA did not accumulate in the NAH or TL of either adult or infant guinea Tables III and IV report sodium concentration in plasma, NAH and TL MSG or NaCl given by gavage to adult and infant guinea pigs respectively. change in sodium levels was observed in adult guinea pigs, but in S-day-old animals 180 min after NaCl administration, sodium increased in the NAH, MSG had no effect on plasma, NAH or TL sodium concentrations.
pigs. after No Qral
316
No GA accumulation was observed in the NM of either adult or infant guinea pigs after oral admin~t~tion of MSG at doses reportedly neurotaxic for 3-day-old animals 13, 41. The present results parallel those previously reported on adult and infant rats [1] and suggest that it may not be necessary for GA levels to be increased in discrete brain areas for neuronal lesions to occur. The mechanism of neurotoxicity suggested for mice cannot be generalized. In this study, as in fl]_ we cannot ascribe the neurotoxic effect of MSG to its sodium content, since no change was observed in sodium levels in NAH and TL of either adult or infant guinea pigs after oral MSG.
The authors wish to thank Dr. A. ~av~la~o and Mr. M. Fittipalcti (Chemical Department of the Laboratorio Provinciale di Igiene e Profilassi, Milan, Italy} for kindly providing the facilities and technical assistance for sodium determinations. MSG was kindly supplied by the Ajinomoto Company. REFERENCES 1 L, Airofdi, M. Salmona, P. Ghe22i and S. Garattini, Ghztamic acid and sodium IevcIs in the nucleus arcuatus of the hypoth~~u& of adult and infant rats after oral monosodium glutamate, Toxicol. Lett., 3 (1979) 121--126. 2 V.J. Perez and J.W. Gfney, A~~urnu~at~onof glutamic acid in the arcuate nucleus of the hypothalamus of the infant mouse following subcutaneous ad~n~tratj~n of mo~~od~urn gfutamate, J. Neurochem., 19 (1972) 3777--1782, 3 J.W. Ulney, Q.L. Ho, V. Rhee and T. DeGubareff, Neurotoxic effects of ~~~t~~~t~, N. En& 3, Med,, 289 (1973) 1374-1376. 4 R. Heywood and A.N. Worden, Glutamate toxicity in laboratory animals, in L.J. Filer Jr., S. Garattini, MR. Kare, W.A. Reynolds and R.J. Wurtman (Eds.), Glutamic Acid: Advances in Biochemistry and Physiology, Raven Press, New York, 1979, pp. 263-215. 5 T.J. Luparello, Stereotaxic Atlas of the Forebrain of the Guinea Pig, Karger, Baael, 1967. 6 M. Palkovits, Isolated removal of hypothallunic or other nuclei of the rat, Brain Ras., 59 (1973) 44Q-466. 7 R.L. Young and U.H. Lowry, Quantitative methods for measuring the histocbemical distribution of a&nine, glutamate and glutamine in brain, J. Neurocheti., 13 (1966) 786-793. 8 M.M Bradford, A rapid and sensitive method far the quantjtat~on of rn~~o~ q~~~tit~a~ of protein utilizing the prineipfe of protein-dye binding, And. 3iochem., 72 (1976) 248-254.