J O U R N A L OF APPLIED TOXICOLOGY. VOL. 16(6), 469473 (1996)

Changes in Brain and Plasma Amino Acids of Mice Intoxicated with Methyl Isocyanate

Meenakshi Gupta' and Vijay Prabha' ' Depar~ment o f Chemistry. College 0 1 ' Science. Rochester Institute o f Technology. 85 Lomh Mcrnorial Drive. Rochester. N Y 11623. LISA 'Deparlmciil 01' Microhiology, Panjnh University, Chandignrh I6001 4. Indin

Kcy uords: .*wiiio acid\: HPLC; MIC; neurotoxicity.

Precolumn derivatization with OPA was used f o r t he analysis of brain a n d plasma free amino acids of mice af ter the administration of different doses (0.25 ms0, 0.5 ims0 and LD,,,) of methyl isocyanate (MIC) for different durations (45 min. 4 h, 4 days and 7 days). In general, there were a dose-dependent decrease in brain free amino acid content with the exception of glycine and arginine (increased above the control level with 0.25 m5,, and 0.5 m5,, doses), and taurine which increased with 0.5 I&, and m5,, doses in 45 min. All the amino acids from plasnia were increased with all the three doses, with the exception of arginine which decreased at the 0.25 tm5" dose in 45 min. With increase in duration of observation to 4 h, 4 days and 7 days, the brain amino acid content was still below the control levels and plasma levels were higher as compared to the respective controls. The only exceptions were serine, histidine, alanine and arginine. which decreased on the 7th day. This study suggests that MIC produced an imbalance of both the brain and plasma amino acids, suggesting neurotoxic and systemic effects.

INTRODUCTION

Methyl isocyanate (MIC) is the basic starting material for the manufacture of insecticides like Sevin. Our earlier studies have shown that both of these com- pounds can affect the hepatic drug metabolizing enzymes,' affect the membrane integrity of mice brain synaptosomes and cause a slow shut down of the GABA system (unpublished data). Ultrastructural and pathobiochemicul evidence has established that MIC is capable of causing damage to the tracheo-bronchial epithelium following pulmonary alveolar It has also been shown to affect the reproductive system'.' and CNS involvement is in the form of stroke. encephalopathy" and persistent depression mixed with anxiety.' Animal studies also show the effect of MIC on the nervous system.x Anderson rt (11." have shown that MIC was toxic to brain cells in a dose-depen- dent manner.

Since certain amino acids like excitatory amino acids ( glutamate and aspartate) cause membrane depolariz- ation and increase permeability of cellular membranes to sodium ions,"' and inhibitory amino acid-like GABA has excitatory and depolarizing actions,] i t was thought pertinent to study the effect of MIC on the free amino acids of brain and plasma.

of acyl azide, as described previously. ' Absolute nujol (paraffin oil) was purchased locally. Unless stated otherwise, solutions of substrates and reagents were made in double-distilled water. Stock MIC was diluted in absolute nujol immediately before use.

Animals Adult LACA mice (25-30 g) of either sex were obtained from the University animal house and were housed in plastic cages of dimensions of 29.2 x 19 x 12.7 cm in groups of six. They were given pelleted diet from Hindustan Lever Ltd., tapwater in 250-in1 plastic bottles and maintained at a temperature of 23.5 k 2°C. All animal treatments were made hy intraperitoneal (i.p,) injection. Control group animals were given the vehicle (8 ml kg-' absolute nujol). Blood was drawn from the eye of mice using h e p arinized capillaries into heparinized tubes. Then the animals were sacrificed by decapitation and the brains were removed by surgery.

Experimental design For the experiment. seven groups of mice (six animals per group) were used. The doses and time of sacrifice after administration of MIC are given i n Table 1.

METHODS

Materials

Substrates. chemicals and biochemicals for the assays were purchased from Sigma Chemical Co., St. Louis, MO. Due to the unavailability of MIC in the market, i t was prepared in the laboratory by Curtins degradation

Determination of amino acids by HPLC Brain and plasma free amino acids were extracted. derivatized and estimated according to the method of Rajendra" with slight modification as described earl- ier.13

Statistical analysis All data were analysed by Student's t-test" for statisti- cal differences between the control and test groups. A

470 MEENAKSHI GUPTA AND VIJAY PRABHA

Table 1. Doses of MIC and time of sacrifice for the seven groups of mice ( 1 1 = 6)

0.25 LD,,, dose led to a decrease i n arginine concen- tration in plasma (Table 3).

Dose (pmol kg ' body wt.) and schedule of administration administration

Time of sacrifice after MIC

0.25 LD,, (293.0). single i.p. 0.25 LO^^ (293.0). single i.p. once daily for three consecutive days 0.5 LD,,, (584.0), single i.p. L D , ~ (1170.0), single i.p.

45 min, 4 h and 7th day 4th day

45 min and 4 h 45 min

Time-related changes

As the duration of observation after the i.p. injection of MIC was increased to 4 h, 4 days and 7 days, the brain amino acid content was still below the control level (Table4). Again the plasma amino acids showed an opposite pattern, with the exception of histidine, serine, alanine and arginine, which were found to be decreased on the 7th day of MIC administration (Table 5).

difference between two means was considered signifi- cant with P < 0.05. P < 0.01 and P < 0.001.

RESULTS

Dose-related changes

All the 18 free amino acids from brain (Table 2) were significantly decreased in a dose-dcpendent manner below the control level. with the exception of glycine and arginine which increased above the control level with 0.25 I - I ) ~ , , and 0.5 L I ) ~ , ) doses, and taurine which increased with 0.5 1 . ~ ~ ~ ) and L I ) ~ , ) in 45 min. On the other hand, all the amino acids from plasma were increased with all three doses of MIC. Only the

DISCUSSION

The results of the study show that following an intra- peritoneal injection of MIC to mice there was in general a dose-related decrease in a large number of amino acids in the brain and an increase in the corre- sponding plasma concentrations. The effect could be a cytotoxic effect due to cell membrane injury, causing the leakage of amino acids from brain to blood. It is known that despite its high reactivity, MIC can traverse cell membranes and reach distant organs, perhaps as a reversible conjugate with glutathione. This conjugation in \i\w produces a reactive S-linked product which displays the potential to carbamylate nucleophilic amino acids.I5 The various systemic toxicities associa- ted with exposure of animals or humans to MIC could therefore be due to release of the isocyanate from its

Table 2. Effect of different doses of MIC on mice brain free amino acids after 45 min of adminis- tration"

Amino acid concentrations after 45 min of the administration of different doses of MIC

Aspartate Glutamate Serine Histidine GI yci n e Threonine Taurine Alanine GABA Arginine Tyrosine Methionine Valine Phenyl alanine lsoleucine Leucine Lysine Ornithine

2.00 f 0.20 2.07 f 0.21 1.33 t 0.15 1.61 fO.10 0.34 f 0.02 0.65 f 0.05 0.10 f 0.03 4.91 f 0.10 3.97 i 0.15 0.18f 0.02 3.47 ? 0.50 2.42 f 0.52 2.35 f 0.41 2.56 f 0.06 2.97 f 0.15 1.40f0.10 4.54? 0.47 5.90 k 0.36

1.25?0.13** 1.48 f 0.15' 1.09 f 0.08' 0.93 _+ 0.07** 0.83 f 0.05*** 0.73 ? 0.1 1 O.lO? 0.02 4.52 f 0.33 3.26 ? 0.25* 0.91 f 0.02*** 3.08 ? 0.32 1.18?0.46* 2.10 f 0.20 2.43f0.11 2.49f 0.16" 1.1Of0.10* 3.17 f 0.15"* 6.08 0.42

1.15f 0.11*** 1.17 f 0.04*** 0.56t 0.08*** 0.86 f 0.05"'" 0.71 +0.03*"* 0.70 f 0.10 0.12 ? 0.10* 2.68 f 0.06*** 1.18?0131*** 0.89 f 0.14*** 2.79 f 0.08" 0.94 _+ 0.38** 0.59 ? 0.24** 2.01 _+ 0.01* 2.49f0.11*** 0.68 f 0.09**" 4.22 ? 0.35"'" 5.07 + 0.25*"

1.04k0.14 1.02 f 0.07 0.20 f 0.03 0.53 t 0.01 0.23 ? 0.03 0.24 ? 0.02 0.15f 0.02 0.65 f 0.05 0.99 f 0.33 0.14_+ 0.01 2.82 i 0.10 0.91 ? 0.03 0.41 ? 0.22 0.47 f 0.12 1.03 ? 0.06 0.44 f 0.09 2.41 f 0.21 3.72 f 0.33

"Values are the mean of three independent experiments. Amino acid content is expressed as pmol g ' weight wet tissue. Significant values compared to respective controls: * P I 0.05, * + P < 0.01 and **" P < 0.001.

CHANGES IN BRAIN AND PLASMA AMINO ACIDS OF MICE INTOXICATED WITH METHYL ISOCYANATE 47 1

Table3. Effect of different doses of MIC on mice plasma free amino acids after 45 min of administration

Amino acid concentrations after 45 min of the administration of different doses of MIC

Amino acid Control 114 LD50 112 LO50 LD50

Aspartate Glutamate Serine Histidine GI ycine Threonine Taurine Alanine GABA Arginine Tyrosine Methionine Valine Phenyl alanine lsoleucine Leucine Lysine Ornithine

50.33 f 1.53 50.00 f 2.00 37.12 f 0.83 39.59 f 0.52 19.87 f 1.03 50.10 f 1.01 16.42 f 0.40 38.40 f 0.50 9.19 f 0.27

124.67f 1.53 145.94 f 1 .OO 16.51 f 0.44

112.31f 1.13 97.41 f 0.52 8.81 f 0.27

31.44 f 0.52 357.38 f 0.54 59.22 f 0.26

19O.OOf 26.63** 290.67 f 10.58*** 51.95 f 5.26**

175.33 f 7.64*** 56.01 f 8.33** 54.90 f 2.23* 40.73 f 5.13**

259.27 f 25.20*** 35.33 f 2.03*** 37.50 f 6.53***

189.56 f 2.52*** 56.15f 11.12** 90.33 f 2.78** 90.58 5 10.02 88.79f 11.10** 64.49 f 0.52***

420.06 f 6.95*** 7.63 f 0.51***

198.00 f 7.64*** 412.67 f 12.59*** 264.74 f 10.00*** 333.69 f 19.02*** 112.01 f 15.23*** 142.07 f 5.51*** 102.92 f 15.00"" 103.24f 10.41*** 227.51 f 20.8*** 250.00 f 14.56*** 335.79 f 7.74*** 169.38 f 12.53*** 180.56 f 13.23*** 155.52 f 1.67*** 89.79 f 3.06*** 91.71 ?6.11***

420.39 f 1.05*** 295.86 k 8.08**

500.67 f 23.07*** 475.00 f 2.00*** 324.89 5 7.77*** 970.41 f 82.25*** 195.67 f 6.56*** 153.14 It 20.2** 123.18 f 7.64*** 19.48 f 0.52***

927.52 f 42.59*** 261.75 f 10.00*** 481.06f 12.59*** 707.13 f 52.83*** 858.39 f 75.67*** 180.50 f 3.420*** 744.9 f 63.21*** 232.10f 12.33*** 500.97f 13.01*** 494.37 f 24.66***

Values are the mean of three independent experiments. Amino acid content is expressed as pmol ml-' plasma. Significant values compared to respective controls: * P < 0.05, **P< 0.01 and ***P< 0.001.

Table 4. Effect of 1/4 LD,, dose of MIC on mice brain free amino acids after different durations of administration"

Amino acid concentration at different durations after administration of MIC

Amino acid Control 4 h 4th day 7th day

Aspartate Glutamate Serine Histidine GI ycine Threonine Taurine Alanine GABA Arginine Tyrosine Methionine Valine Phenyl alanine lsoleucine Leucine Lysine Ornithine

2.00 f 0.20 2.07 f 0.21 1.33 f 0.15 1.61 fO.10 0.34 f 0.02 0.65 f 0.05 0.10 f 0.03 4.91 fO.10 3.97 f 0.15 0.18 f 0.02 3.47 f 0.50 2.42 f 0.52 2.35 f 0.41 2.56f 0.06 2.97 f 0.15 1.40 f 0.1 0 4.54 f 0.10 5.90 f 0.36

0.92 f 0.02 0.73 f 0.02** 1.18f0.15 0.50 f 0.02*** 0.12 f 0.01*** 0.59 f 0.04 0.13 f 0.02 0.61 f 0.02*** 1.51 f 0.04*** 0.11 f 0.02* 0.91 f 0.04** 1.24 f 0.21 * 0.90 f 0.01*** 0.86 ? 0.03*** 1.05 f 0.13*** 0.82 f 0.03** 0.85 f 0.03*** 0.88 f 0.02***

0.61 f 0.02** 0.50f 0.04** 0.63 f 0.03** 0.31 It 0.02*** 0.10 f 0.04** 0.42 f 0.04** 0.06 f 0.01* 0.48 f 0.02*** 2.09 f 0.12*** 0.13f 0.02" 1.91 f 0.03** 1.51 fO.17* 1.43 f 0.03* 0.91 f 0.04*** 0.07 f 0.01*** 0.11 *0.03*** 2.15f 0.11*** 2.48 f 0.10***

0.90 f 0.04** 0.98 f 0.03** 1.07 f 0.08' 0.80 f 0.02*** 0.30 f 0.01* 0.61 f 0.02 0.1 1 f 0.02 0.69 f 0.03*** 3.92 f 0.08 0.20 f 0.02 1.96 f 0.07** 2.03 f 0.08 1.96 f 0.04 2.30 f 0.10* 1.23 f 0.20** 3.69 f 0.17*** 5.02 f 0.28' 5.00 f 0.26'

"Values are the mean of three independent experiments. Amino acid content is expressed as pmol g-' weight wet tissue. Significant values compared to respective controls: * P < 0.001, **P< 0.01 and * * *P< 0.05.

glutathione conjugate, which thus may serve as a vehicle for the transport of MIC in ~ i v o . ' ~ Radioactivity studies have shown the presence of MIC in brain even on day 10.''

The excitatory amino acids (EAAs) L-glutamate and L-aspartate are the most abundant amino acids in brain and play a number of roles in maintaining neural functions. Among these are their use as protein con- stituents, as key intermediates in ammonia metabolism

and as precursors for other neurotransmitters. These transmitters are likely to be involved in virtually all CNS functions, with abnormalities in neurotransmission contributing to the symptoms of a host of neurologi- cal'x and psychiatric disorders. l 9 Glutamate itself serves vitally important functions in the CNS but has enor- mous neurodestructive potential. N-Methyl- aspar art ate (NMDA) glutamate receptor antagonists protect many neurons against glutamate neurotoxicity, while injuring

472 MEENAKSHI GUPTA A N D VlJAY PRARHA

Table 5. Effect of 1/4 L D ~ , ) dose o f MIC on mice p1nsmi.i free amino acids after different duration4 o f administration.'

Amino acid concentration at different durations after administration MIC

Amino acid Control 4 h 4th day 7th day

Aspa rtate Glutamate Serine Histidine Glycine Threonine Taurine Alanine GABA Arginine Tyrosine Methionine Valine Phenyl alanine lsoleucine Leucine Lysine Ornithine

50.33 k 1.53 50.00 f 2.00 37.12 f 0.83 39.59 f 0.52 19.87 f 1.03 50.10f 1.01 16.42 f 0.40 38.40 f 0.50 9.19 f 0.27

124.67 f 1.53 145.94f 1.00 16.51 f 0.44

112.31 f 1.13 97.41 +_ 0.52 8.81 f 0.27

31.44 f 0.52 357.38 f 0.54 59.22 t 0.26

173.33f 15.28* 116.88 k 5.1 2* 209.07 f 14.19* 14.02 -t 0.64** 10.85 f 0.22*** 10.40 f 0.56* 77.26 f 8.01 * 35.67 f 6.03 12.50f 1.50**' 85.83 f 5.20" 79.01 f 3.00* 79.04f 3.34" 24.64 f 4.15* 95.82 f 5.20 13.47 f 0.50** 36.10+3.57***

303.72 f 20.73* 59.73 f 3.52

145.12 i 5.00* 117.87 f 6.55" 41.12 f 3.56**

300.11 f 11.50" 46.02 f 3.50* 54.69 f 4.20 42.06 f 2.61' 76.59 f 4.12* 10.94 f 1 .OO

142.20 f 7.50* 262.50f 12.00" 31.0Ok4.5lx

114.52 f 10.44 174.99 k 14.50* 15.18 f 1.00* 64.48 i 3.12*

379.19 f 17.37** 65.18f 6.02**

51.33 i 4.16 50.00 k 1.80 29.07 f 3.00** 16.45 k 0.51* 35.00 f 5.00" 17.50 f 0.70* 16.23f 0.75 35.06f 2.00*** 12.83f 1.04** 62.42 f 3.17*

148.28f 10.51 17.59 f 2.50

11 5.84 k 6.60 112.07 f 7.54 12.41 f 0.52** 31.33k3.51

344.04 f 38.96 61.73 f 4.03

"Values are the mean of three independent experiments. Amino acid content is expressed as +mol m l ' plasma. Significant values compared to respective controls: * P I 0.001, * * P c. 0.01 and ***PI 0.05.

or destroying certain other neurons and inducing psy- chotic symptoms and memory impairment.") I t has also been seen that glutamate decreases intracellular pH (pHi) in cultured rat hippocampal neurons and is symp- tomatic of neurotoxic processes." Thus an inhibition of these EAAs, as also seen in the present study (Tables 2 and 4). might lead to hindrances in nerve transmission and cellular metabolism and cause psychi- atric disturbances.

The observed decrease (Table '7) i n GABA content might be correlated with a decrease i n cholesterol/phospholipid (Ch/PL) ratio and an increase in lipid peroxidation (unpublished data), since it has been shown that GABA uptake is markedly sensitive to reduction in membrane Ch/PL ratio in brain.'" GABA uptake is decreased both with a decrease i n Ch/PL ratio and an increase in lipid/protein ratio. A reduction of GABA below normal in brain induces convulsions and hyperexcitability.'3 Since, along with GABA. most of the amino acids have been shown to be significantly decreased (Tables 2 and 4). these changes could be the cause for the non-survival of the affected animals even for a few days at sublethal doses.

Fekkes et 01.'~ have shown the usefulness of plasma amino acid concentrations in discriminating patients with transient acute polymorphic psychoses ( APP) characterized by psychosensory symptoms. They have

also shown abnornial plasma levels 9f serine, methion- ine and taurine in APP (as is also observed in the present study; Tables 3 and 5 ) and that the determi- nation of the serine tyrosine-serine-methionine ratio may be a useful diagnostic validator in a group of patients with APP.

Alterations in the concentrations of aromatic amino acids (AAAs) phenyl alanine and tryosine may lead to disturbances of inonoamine neurotransmitter in brain. Such changes could play a role i n the pathogenesis of hepatic encephalopathy.'5 Disturbances in sulphur amino acids have also been shown to be associated with psychiatric or neurological symptomatology.'"

Thus i t could be said that MIC is a potential neuro- toxicant and has systemic effects. The findings of this study in the light of the above discussion suggest that some of the neurological symptoms. like depression, anxiety, restlessness and neurosis," as seen in the Bhopal survivors might also be due to the imbalance of the amino acids caused by MIC.

Acknowledgement

This work i s part of ;i PhD thesis acccpred at Panjah University. Chandigarh 160014. India.

CHANGES IN BRAIN AND PLASMA AMINO ACIDS OF MICE INTOXICATED WITH METHYL ISOCYANATE 473

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