Journal of Neurwhemistr? Raven Ress, Ltd., New York Q 1990 Internattonal Society for Neurockmistry

Neurotransmitter Changes in Guinea-pig Brain Regions Following Soman Intoxication Paul Fosbraey, Janet R. Wetherell, and Mary C. French Biology Division, Chemical Defence Establishment, Porton Down, Salisbury, Wiltshire, England

Abstract: The effects of the organophosphate acetylcholinesterax (AChE) inhibitor soman (31.2 pgkg s.c.) on guineapig brain AChE, transmitter, and metabolite levels were investigated. Concentrations of acetylcholine (ACh) and choline (Ch), noradrenaline (NA), dopamine (DA), 5-hydroxytryg tamine (5-HT), and their metabolites, and six putative amino acid transmitters were determined concurrently in six brain regions. The brain AChE activity was maximally inhibited by 90%. The ACh content was elevated in most brain areas by 15 min, remaining at this level throughout the study. This increase reached statistical significance in the cortex, h i p pocampus, and striatum. The Ch level was significantly elevated in most areas by 60-120 min. In all regions, levels of NA were reduced, and levels of DA were maintained, but

those of its metabolites increased. 5-HT levels were unchanged, but those of its metabolites showed a small increase. Changes in levels of amino acids were restricted to those areas where ACh levels were significantly raised: Aspartate levels fell, whereas y-aminobutyric acid levels rose. These findings are consistent with an initial increase in ACh content, resulting in secondary changes in DA and 5-HT turnover and release of NA and excitatory and inhibitory amino acid transmitters. This study can be used as a basis to investigate the effect of toxic agents and their treatments on the different transmitter systems. Key Words: Soman poisoning4uineapig-Transmitter content-CNS. Fosbraey P. et al. Neurotransmitter changes in guinea-pig brain regions following soman intoxication. J. Neurochem. 54, 72-79 (1990).

Organophosphates, such as soman (pinacolyl methylphosphonofluoridate), act by inhibiting acetylcholinesterase (AChE) both peripherally and centrally, with a subsequent accumulation of acetylcholine (ACh). Studies have shown that soman, diisopropylphosphofluoridate (DFP), and paraoxon produce a regionally variable elevation of ACh and choline (Ch) levels in the rat brain within minutes; this elevation is associated with a peak brain inhibition of >90% (Wecker and Dettbarn, 1979; Shih, 1982; Lundy and Shih, 1983; Potter et al., 1985; Rynn and Wecker, 1986). ACh levels in rat brain start to return to normal values, whereas the brain AChE content is still >80%inhibited (Wecker et al., 1977; Shih, 1982; Hoskins et al., 1986). Also, rats showed complete physical and functional recovery 12 and 24 h after injection of sublethal and lethal doses of soman when blood, brain, and diaphragm cholinesterase was still 70-80% inhibited (Jovic, 1973). A possible explanation for this observation is that complex interactions with other transmitter systems result

in a compensatory feedback control of ACh levels despite the inhibition of AChE, enabling a finely balanced control of neuronal function. Therefore, it is conceivable that major changes in the concentration of ACh resulting from inhibition of AChE could result in corresponding, compensatory changes in other transmitter systems. In previous studies of the effects of AChE inhibitors on noncholinergic neurotransmitters, levels have been measured either in the brain as a whole or in a single region such as the striatum, an area rich in cholinergic innervation. Such studies can give useful information as to the extent of interaction between neurones using different transmitters and the relative activity of neuronal groups using the same transmitter in different brain regions. However, no study has sought a comprehensive evaluation of AChE inhibitors on all the major transmitter systems, instead, studies have concentrated on single transmitters. In this way, the levels of 5-hydroxytryptamine (5-HT)and its chief metabolite

Received March 16, 1989; revised manuscript received May 12, 1989; awepted May 22, 1989. Address correspondence and reprint requests to Dr.P. Fosbraey at Biology Division,Chemical Defence Establishment, Porton Down, Salisbury, Wiltshire SP4 OJQ, U.K.

Abbreviations used: ACh, acetylcholine;AChE, acetylcholinesterase; Ch, choline; DA, dopamine; DFP, diisopropylphosphofluoridate; WPAC, 3,4-dihydroxyphenylaceticacid GABA, y-aminobutyric acid; 5-HIAA, 5-hydroxyindoleaceticacid; 5-HT, 5-hydroxytryptamine; HVA, homovanillic acid; NA, noradrenaline; Tau, taurine.

72

BRAIN TRANSMITTER CHANGES AFTER SOMAN

5-hydroxyindoleacetic acid (5-HIAA) were shown to rise following administration of soman in the rat (Prioux-Guyonneau et al., 1982) and DFP in the rabbit (Barnes et al., 1975). The effects of AChE inhibitors on monoamine levels in the brain are more confusing: DFP caused a significant reduction in noradrenaline (NA) and elevation of dopamine (DA) levels in rabbit brain (Glisson et al., 1974), whereas in the rat it produced no change in DA levels but an increase in content of DA metabolites (Fernando et al., 1986). Changes in the content of putative transmitter amino acids following AChE inhibition have also been observed (CoudrayLucas et al., 1984; Ho et al., 1984). Most studies of brain neurochemistry following organophosphate intoxication have been performed in the rat; however, Inns and Leadbeater (1983) have suggested that the guinea-pig is a better model for predicting the efficacy of treatments for organophosphate poisoning in primate species. The present study used six brain regions of the guinea-pig for the concomitant measurement of the neurochemical changes in different transmitter systems at various intervals following exposure to a dose of soman that killed all guinea-pigs in 24 h. In this way, a comprehensive assessment of the primary and subsequent events in the neurochemical adjustment to AChE inhibition and its relation to soman toxicity was made in a more relevant species. MATERIALS AND METHODS Materials Microperoxidase (EC 1.1 1.1.7; sodium salt from equine heart cytochrome c), choline oxidase (EC 1.1.3.17; from Alcaligenes species), and AChE (EC 3. l. l .7; type V-S from electric eel) were from Sigma Chemical Co., Ltd. Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione)was from LKB Wallac. Glycine, phosphate buffer salts, acetylthiocholine, dithionitrobenzoic acid, and sodium periodate were from BDH Chemicals, Ltd. All HPLC chemicals and water of HPLC grade were also from BDH.

Animals and tissue dissection Male Dunkin-Hartley guinea-pigs (weighing 250-280 g) were injected with soman (3 1.2 pdkg s.c.) or 0.9% saline (1 ml/kg; controls) and killed by exposure to microwave radiation (2.45 GHz, 2.4 kW, 7 s; Elnode EF-R microwave applicator) after 15, 30, 60, 120, and 240 min (controls after 30 min only). The animals were then decapitated, and their heads were immersed in liquid nitrogen for 15 s to cool the tissue. The brain was removed from the skull and dissected into regions as described by Glowinski and Iversen (1 966); the hypothalamus was not, however, separated from the midbrain. Brain regions were sectioned in the midsagittal plane, and left and right hemiregions were used alternately to quantify ACh and Ch, and biogenic amines and transmitter amino acids.

Assay of ACh and Ch Brain areas were homogenised in 15% formic acid in acetone (100 mdml) for 30 s and left to stand on ice for 30 min. After centrifugation (10,000 g for 10 min), 100 pl of supernatant was evaporated at 95°C (5-10 min), the residue was

73

resuspended in 100 pl of water, and 10 pl of 0.5% (wt/vol) sodium metaperiodate was added. After 20 min, 900 pl of water was added, and the samples were stored at -20°C until assay. Samples were assayed on a LKB automated luminometer model 1251 as previously described (Wetherell et al., 1989). The reaction medium consisted of 0.05 M glycine buffer (pH 9.0) containing 7.5 pglml of microperoxidase and 10 pM luminol. This mixture was left to stand for 10 min. One hundred microlitres of sample was added to 900 pl of reaction medium in duplicate, 10 pl of a standard solution of Ch and ACh (20 pmol) was added to the second tube. The samples were placed in the luminometer at 25°C and left for 30 min before 10 pl of choline oxidase (10 units/ml) was added. The peak height of light emission (mV) for Ch was recorded over a 2-min period. Ten microlitres of AChE (80 units/ml) was added 50 min later for quantification of ACh. Standard curves for Ch and ACh ( 10-60 pmol) were camed out at the start of each day to check the linearity of the reactions. The concentration of Ch and ACh in the samples was calculated from the difference in the light emission measured in the presence and absence of 20 pmol of Ch and ACh as a standard. The extraction efficiency was 93% for Ch and 85% for ACh. Results were expressed as mean & SEM values (in nanomoles per gram).

Assay of biogenic amines Tissue samples were homogenised ( I 5 s, 100 mg/ml) in 0.1 M perchlonc acid containing 3,4dihyroxybenzylamine (0.5 p M ) and DL-homoserine ( 1 mM) as internal standards. After centrifugation (10,000 g for 10 min), 20 pl of the supernatant was analysed by reversed-phase HPLC with electrochemical detection as previously described (Wetherell et al., 1989). The mobile phase consisted of 120 ml of I M KH2P04,680 ml of water, 3 ml of EDTA (10%wt/vol), 140 ml of methanol, and 100 mg of I-octanesulphonic acid (sodium salt) adjusted to pH 3.2 with orthophosphoric acid. Tissue levels were determined by means of internal and external standards and expressed in terms of nanomoles per gram of tissue; percentage recovery ranged between 85 and 97%. Results were expressed as mean ? SEM values.

Assay of amino acids Twenty microlitres of the perchloric acid tissue supernatant was neutralised and diluted with 0.98 ml of sodium tetraborate (0.1 M, pH 9.5). The amino acids Asp, Glu, Gln, Gly, taurine (Tau), and y-aminobutyric acid (GABA) were assayed by isocratic HPLC with electrochemical detection following derivatisation using u-phthalaldehyde/2-methyl-2propane thiol derivatising reagent as previously described (Wetherellet al., 1989).Forty microlitres ofsample was mixed with 10 pl of reagent by means of a Gilson 23 1-40I autosampling injector and left to react for 2 min before 20 pl was injected onto a MicroSpher C18 column (50- X 4.5-mm stainless steel cartridge; particle size, 3 pm: Chrompak UK. Ltd.). The mobile phase, pumped at a flow rate of 2.5 ml/ min, consisted of 60 ml of K2HPOI ( I M ) , 2.2 ml of EDTA (10% wt/vol), 520 ml of water. 420 ml of methanol, and 40 ml of tetrahydrofuran; the pH was adjusted to 7.0 with NaOH. Tissue levels were determined by means of internal and external standards and expressed in terms of micromoles per gram of tissue; percentage recovery ranged between 80 and 105%. Results were expressed as mean k SEM values.

Assay of AChE Groups of four or five guinea-pigs were injected with soman (31.2 p g k g s.c.) or 0.9% saline ( I ml/kg; controls) and killed J Neurochem, Vol 54. No 1. 1990

P. FOSBRAEY ET AL.

74

by cervical dislocation at 15, 30, 120, and 240 min. The animals were decapitated, and the brains were dissected into regions as described previously but without hemisection. Brain regions were homogenised (cortex, 200 mg/ml; stnaturn, 50 rng/rnl; other regions, 100 mgjml) in ice-cold 0.1 M phosphate buffer at pH 8.0 for 15-30 s. Samples were stored at 4°C until assay. AChE was quantified using a modified method of Ellman et al. (1961). Samples were diluted with 0.1 M phosphate buffer (pH 8.0) (striaturn, 150; cortex, 1: 200; other regions, I: 100) and assayed at 30°C using 1 .O mM acetylthiocholine on a Pye Unicam PU 8800 UV/VIS spectrophotometer. Assays were done for 10 rnin and were linear throughout this time. Control results were expressed as micromoles of acetylthiocholine hydrolysed per minute per gram wet weight of tissue. Test results were expressed as percentage inhibition of the control results.

RESULTS Signs of poisoning Following injection of 10 guinea-pigs with soman, the earliest signs of poisoning were seen within 15 min: These consisted of hyperactivity, tremor, and chewing, progressing to marked salivation, lachrymation, urination, and defaecation. After 30-90 min, coordination of locomotor activity became disturbed, convulsions were evident, and death resulted from respiratory failure after 4-24 h. In subsequent experiments, all animals were killed within 4 h.

Regional brain AChE AChE was inhibited by >60% in all brain areas except the striatum (45% inhibition) by 15 rnin (Table I). Inhibition was maximal at 30 rnin (-90% in all areas) and was maintained at this level until 120 min, followed by a slight recovery (4-1 1%) of enzyme activity by 240 min.

ACh and Ch ACh. There was an increase in the content of ACh in all brain areas, except the medulla-pons, 15 rnin after soman injection (Table 1). This increase was statistically significant in the cortex, hippocampus, and striatum, where the ACh content showed a maximal increase of 66, 37, and 58%, respectively, within 60120 min, and levels remained elevated for 4 h. A significant increase was observed in the midbrain (23%) by 120 min, but the increase of 43% observed in the cerebellum was not significantly different from control levels. Ch. There was an increase in Ch content in all brain areas by 15-30 rnin except the cortex, which showed a reduction in content at 15 min. Ch levels showed a maximal increase of 30-70% in all areas at 120 min and was statistically significant at a minimum of one time point in the study in all regions except the medulla-pons. Levels were returning to control values in most areas by 240 min.

TABLE 1. Eflect of soman on brain levels of ACh and Ch and inhibition of AChE Marker, time in rnin (n)

Cerebellum

Hippocampus

Medulla-pons

Cortex

Midbrain

Striatum

4.05 k 0.36

5.27 S 1.18 4.14k 0.61 4.08 f 0.70 5.79 k 0.86 5.12 % 0.83

19.62 f 0.87 24.30 t 0.75" 25.90 f 1.65' 24.40k 1.36' 26.90 k 1.63" 24.40 t 1.69'

21.50 k 1.13 22.30 1.74 19.60 t 1.85 23.20f 2.19 23.40 t 2.92 19.60 t 1.50

13.20 k 0.47 18.40 f 1.15" 19.10 t 1.06" 21.90 t 2.21" 20.10 t 1.70" 20.20 t 2.03

28.85 f 1.1 1 32.40 5 1.55 30.70 t 2.56 31.20 f 1.87 35.50 t 2.89' 34.70 f 2.99

34.20 k 1.62 45.90 t 2.9gb 43.30 f 3.29' 41.90 t 3.08' 54.00 t 3.55" 42.lOt 1.89'

5 I .60 k 2.69 63.00 f 6.54 57.30 k 4.06 66.50 k 5.30' 86.50 2 13.70' 63.80 k 6.24'

37.80 f 2.73 38.80 f 2.20 40.40 f 2.39 47.10 t 4.98 53.95 f 5.86' 45.20 f 4.70

45.10 f 3.17 5 1.20 t 2.89 54.80 f 6.20 56.20 t 6.52 60.30 t 9.40 53.20 f 6.07

39.90 f 2.91 34.60 t 3.30 41.50 f 3.34 42.00k 3.16 53.30t 5.17' 38.30 ? 1.39

46.90 t 3.46 49.40 f 3.60 49.60 t 4.32 61.90f 5.94' 64.80 ? 7.29' 57.60 f 3.13'

42.70 f 1.91 44.90 t 4.50 48.60 f 2.50' 49.80 t 2.8 I 59.10 7.09' 48.50 k 5.20

12.27 k 0.57

6.05 t 0.57

9.25 t 0.24

5.72

9.32 ? 0.16

21.30 S 0.53

0.0 73.5 k 3.0 93.3 k 3.1 93.9 _+ 1.3 90.4 2.0

0 .o 68.7 f 3.1 90.9 f 2.4 9 1 . 9 t 1.8 82.2 f 4.1

0.0 64.7 t 2.1 92.1 t 3.7 90.0 t 1.7 80.6 t 3.9

0.0 70.3 f 1.2 88.3 t 5.0 89.2 t 2.2 83.6 t 2.9

0.0 72.5 f 3.2 93.6 5 2.9 92.9 t 0.5 87.3 t 1.3

0.0 44.7 5.83 87.6 t 6.30 92.4 t 2.06 81.6f 3.46

ACh 0 (26) 15 (8) 30 (9) 60(11) 120 ( I I ) 240 (7)

*

'

Ch 0 (26) 15 (8)

30 (9) 60(11) 120 ( I I ) 240 (7)

*

AChE activity Basal

W inhibition 0 IS 30 I20 240

*

+_

0.18

*

Guinea-pigs used for determination of ACh and Ch levels (nmol/g wet weight of tissue) were killed by microwave irradiation (7 s) at fixed intervals after administration of soman (31.2 pglkg s.c.). Brain AChE activity (pmol/min/g of tissue) was determined in animals killed by cervical dislocation. Basal activity was determined in control animals, which formed the basis for calculation of percentage inhibition of the enzyme in animals treated with soman. Data are mean S SE values. A statistically significant difference from control values is indicated as follows: " p < 0.001, ' p < 0.01, ' p < 0.05. J. Neurochen.. Val. 54, No. 1. 1990

BRAIN TRANSMITTER CHANGES AFTER SOMAN

Biogenic amines NA. There was a statistically significant reduction in NA content in all regions following soman administration (Table 2). The change was least marked in the striaturn, the region with the lowest concentration of NA. In all other regions, the reduction in content was significant within 1 h and increased with time. Change in content, expressed as a percentage of the control value, was most marked in the cerebellum, cortex, and hippocampus, with an approximate maximal 60% reduction. Decreases in the medulla-pons and midbrainhypothalamus, the regions of highest NA content, were

75

40%. The decline in content appeared to plateau between 2 and 4 h. DA and metabolites. There was no significant change in the content of DA in the hippocampus and cortex. However, there was a moderate increase in DA level in the cerebellum, medulla-pons, and midbrain-hypothalamus, which was statistically significant by 60 min and a transient increase in the striaturn (Table 2). Levels of both 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) increased markedly with time following soman administration in all brain regions, with DOPAC levels being statistically

TABLE 2. Eflect of soman on brain levels of biogenic amines Marker, time in min (n)

Cerebellum

Hippocampus

Medulla-pons

Cortex

Midbrain

Striatum

0.98 f 0.08 0.83 f 0.06 0.66 t 0.05' 0.58 t 0.07 0.49 f 0.06' 0.52 t 0.07'

'

1.33 t 0.08 1 . 1 4 t 0.06 0.85 ? 0.08' 0.76 f 0.08' 0.60 t 0.06' 0.56 ? 0.04'

2.59 t 0.22 2.58 ? 0.33 1.75 ? 0.15' 2.35 f 0.43 1.58 t 0.17' 1.75 f 0.20'

0.87 t 0.06 0.67 t 0.06" 0.67 t 0.06' 0.47 t 0.05' 0.39 t 0.05' 0.38 t 0.03'

2.40 t 0.09 2.52 t 0.18 2.13 C 0.14 1.86 C 0.15' 1.51 f 0.15' 1.50 -C 0.08'

0.59 f 0.06 0.44 t 0.08 0.5 I f 0.03 0.49 f 0.03 0.44 2 0.04" 0.50 f 0.05

0 (17) 15 (7) 30 (9) 60 ( l o ) 120 (8) 240 (8)

0.12 _+ 0.01 0.14 f 0.02 0.13 f 0.01 0.16 f 0.01 0.19 f 0.02" 0. I7 _+ 0.02"

0.79 f 0.1 I 0.88 t 0.16 0.63 t 0.08 1.03 t0.19 0.82 t 0. I6 0.60 t 0.08

0.26 f 0.02 0.35 t 0.07 0.29 t 0.03 0.37 t 0.03' 0.41 f 0.04' 0.44 t 0.04'

2.08 f 0.24 1.81 t 0.32 2.02 f 0.13 2.00 2 0.13 2.01 t 0.11 2.10 t 0.25

1.1 I C 0.05 1.45 f 0.17 I .22 & 0.05 1.45 2 0.08' 1.71 k 0.25" 1.41 2 O.Ogb

42.82 f 1.22 43.68 ? 3. I8 38.02 2.91 54.32 2.19' 48.31 f 2.95 35.51 t 2.70"

WPAC o (17) 15 (7) 30 (9) 60 (10) 120 (8) 240 (8)

0.09 f 0.01 0.07 f 0.02 0.10 f 0.01 0.18 f 0.03' 0.23 f 0.04' 0.24 & 0.03'

0.22 t 0.03 0.24 t 0.03 0.29 f 0.02" 0.61 f 0.15" 0.71 t 0.12' 0.81 ? 0.07'

0.26 t 0.02 0.27 f 0.03 0.33 t 0.04 0.52 f 0.09' 0.80 tO.13' 0.83 f 0.14'

0.47 f 0.04 0.57 ? 0.05 0.74 f 0.04' 0.92 t 0.12' 1.31 f 0.16' 1.68 t 0.23'

0.45 & 0.02 0.58 k 0.05" 0.72 & 0.03' 1.62 k 0.19' 1.50 f 0.14'

5.02 t 0.16 6.10? 0.51 6.24 f 0.53" 9.49 t 1.02' 15.11 f2.08' 14.08? 1.27'

0.16 0.01 0.17 t 0.02 0.16 rt: 0.01 0.29 t 0.03' 0.43 _+ 0.06' 0.58 t 0.07'

0.33 f 0.03 0.38 t 0.06 0.34 f 0.03 0.66 t 0. I I 0.97 ? 0.14' 1.51 t 0.16'

0.32 f 0.02 0.33 t 0.03 0.33 f 0.02 0.58 t 0.08' 0.88 f 0.13' 1.04 t 0.19'

0.82 0.07 0.84 f 0.1 I I .OO t 0.04" 1.31 f 0 . 1 I C 1.76 t 0.16' 3.1 I t 0.31'

0.82 t 0.04 0.93 f 0.07 0.94 t 0.05 I .44 f 0.09' 2.22 & 0.17' 2.80 f 0.25'

7.28 f 0.32 7.67 i 0.83 7.28 t 0.52 ll.16?0.75c 15.60f 1.62' 19.79 ? 2.05'

0.38 A 0.05 0.32 ? 0.05 0.32 t 0.03 0.41 t 0.05 0.49 rt: 0. I I 0.56 t 0.14

1.43 t 0.07 1.43 f 0.14 1.34 t 0.08 1.56 f 0.10 1.54 f 0.08 1.74 k 0.18

2.80 ? 0.23 2.55 f 0.34 2.47 ? 0.33 2.97 t 0.24 3. I2 ? 0.24 3.47 t 0.40

1.38 f 0.04 1.36 ? 0.12 1 .SO 2 0.06 1.57fO.lI 1.45 f 0.06 1.69 ? 0. I I '

3.07 t 0.19 3.30 % 0.31 3.45 r 0.15 3.37 t 0.22 3.40 t 0.21 3.57 f 0. I I

1.14?0.06 1.40 i 0.36 1.07 ? 0.08 1.25 t 0.08 I .09 ? 0.06 1.16_+O.ll

0.17f 0.02 0. I7 t 0.02 0.16 f 0.01 0.21 A 0.02 0.26 ? 0.03" 0.25 t 0.05

0.61 t 0.03 0.61 f 0.04 0.62 t 0.02 0.74 f 0.06 0.85 t 0.05' 0.98 f 0.13'

0.82 t 0.07 0.69 t 0.07 0.72 f 0.1 1 0.90 t 0.08 1.01 f 0.12 1.16 t 0.22

0.45 ? 0.02 0.40 A 0.03 0.50 t 0.03 0.56 t 0.05 0.62 f 0.04' 0.81 t 0.09'

1.15 ? 0.05 1.06 f 0.08 1.23 t 0.07 1.28 f 0.13 1.46 O.IOb 1.55 t 0.16"

0.57 ? 0.03 0.51 2 0.06 0.55 ? 0.03 0.69 _+ 0.07 0.73 ? 0.04' 0.91 ? O . I l h

NA

o (17) 15 (7) 30 (9) 60 (lo) 120 (8) 240 (8)

DA

HVA

o (17)

15 (7) 30 (9) 60 (10) 120 (8) 240 (8)

'

*

5-HT

0 117) 15 (7) 30 (9) 60 (10) 120 (8) 240 (8) 5-HIAA o (17) 15 (7) 30 (91 60 (10) 120 (8) 240 (8)

'

1.11 f O . I l C

+_

*

*

All guinea-pigs were killed by microwave irradiation (7 s) at fixed intervals after administration of soman ( 3 1.2 WeJkg s.c.). Data are mean t SE values (in nmol/g wet weight of tissue). A statistically significant difference from control values is indicated as follows: "p < 0.05, ' p < 0.01, ' p < O.Oo1.

J Neurochem , Yo1 54,

No 1. 1990

P. FOSBRAEY ET AL.

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significantly different from the control value within 30 min and HVA levels by 60 min. Metabolite/DA ratios showed the greatest increase in the hippocampus, with less marked changes in the cortex, midbrain-hypothalamus, and striaturn and minor changes in the cerebellum and medulla-pons. The DOPAC/HVA ratio showed a transient increase (30-120 min) in all areas except the cerebellum and medulla-pons. 5-HTand metabolite. There was no change in the levels of 5-HT in any brain region, whereas 5-HIAA levels increased with time in all areas, becoming statistically significantly different from the control value at 120 min, except in the cerebellum and medulla-

pons (Table 2). The 5-HIAA/S-HT ratio only showed a statistically significant increase in the hippocampus, cortex, and striatum. Amino acids Asp, Glu, and Gln. A decrease in Asp levels was detectable in the cortex, hippocampus, and striatum 60 min after soman administration, with no change in the remaining regions (Table 3). No change in Glu levels was detectable in any brain region. Gln levels were significantly raised by 120 min after soman administration but had returned to control values by 240 min. Giy, Tau, and GABA. The levels of Gly and Tau

TABLE 3. Effect of soman on brain levels of transmitter amino acids Marker, time in min (n)

Cerebellum

Hippocampus

Medulla-pons

Cortex

Midbrain

Striatum

2.45 f 0.31 2.04 f 0.27 2.01 f 0.07 2.04 f 0.19 1.99 f 0.24 2.39 t_ 0.26

2.69 f 0.33 2.74 f 0.59 1.92 f 0.10 1.50 20.16' 1.50 f 0.49 1.60 t 0.20"

2.65 f 0.41 2.39 t 0.36 3.06 f 0. I3 2.59 f 0.36 2.93 0.37 3.12 t 0.53

3.78 t 0.31 3.54 f 0.58 3.15 f 0.16 1.92 f 0.17' 1.66 f 0.70" 2.02 t 0.47"

3.48 t 0.25 3.55 ? 0.48 3.65 f 0.44 2.55 ? 0.37 2.78 f 0.39 2.54 2 0.18"

2.82 f 0.27 2.42 0.53 2.10 f 0.23 1.47 0.19' 1.35 f 0.47" 1.65 f 0.46

8.63 0.48 9.19 f 0.68 9.43 f 0.47 8.65 f 0.76 8.72 f 0.58 8.56 f 0.46

8.73 f 0.53 8.98 0.59 8.78 f 0.63 8.20 t 0.84 7.16 f 0.51 7.99 t 0.39

5.82 f 0.32 5.61 f 0.25 7.18 f 0.49" 6.99 f 0.70 6.46 f 0.41 5.94 f 0.7 1

10.45 0.58 10.72 f 0.46 9.52 f 0.29 8.53 t 0.48" 8.16 f 0.93 8.59 t 0.70

9.13 f 0.59 9.04 i 0.49 8.88 f 0.77 7.90 i 0.42 8.55 f 0.65 8.07 i 0.46

9.39 f 0.47 9.70 f 0.83 8.66 f 0.50 8.08 f 0.45 8.26 1.32 7.26 f 1.19

4.35 t 0.40 5.59 f 0.74 5.58 f 0.54 5.32 f 0.47 6.41 f 0.42' 3.90 f 0.47

3.95 t 0.28 4.29 f 0.69 4.06 f 0.41 5.59 f 0.48" 6.01 t 0.77" 3.47 f 0.42

2.58 t 0.17 3.08 f 0.17 3.89 f 0.68 3.71 f 0.39" 4.10 f 0.20' 2.45 f 0.53

4.88 f 0.18 5.84 f 0.93 6.18 t 0.85 6.92 f 0.95 7.01 f 0.71" 4.09 f 0.45

4.29 t 0.13 4.79 +- 0.54 5.59 t 0.86 4.65 f 0.58 6.24 2 0.48' 3.50 f 0.52

5.45 f 0.27 5.53 f 0.39 5.55 f 0.42 6.65 5 0.53 7.30 f 0.66" 3.72 f 0.58"

1.08 f 0.19 1.57 f 0.42 I .54 0.26 1.12 t 0.09 1.22 f 0.10 1.25 2 0.26

1.12 5 0.17 1.33 f 0.33 1.46 f 0.20 1.31 f 0.19 1.22 f 0.20 0.90 5 0.06

2.33 f 0.14 2.40 f 0.15 3.23 0.23" 2.72 f 0.37 2.13 fO.10 2.04 t 0.42

1.01 f 0.1 1 1.46 t 0.37 1.55 t 0.36 I .20 f 0.32 1.25 f 0.20 1.00 f 0.16

1.65 k 0.3 1 1.74 f 0.27 1.97 k 0.33 1.48 f 0.18 1.47f 0.10 1.29 f 0.26

1.20 fO.10 1.13 20.36 1.28 f 0.22 1.17 f 0 . 1 5 1.27 e0.15 0.97 f 0.22

2.93 f 0.43 2.80 f 0.79 3.12 f 0.18 2.85 f 0.29 3.19 f 0.21 2.71 f 0.50

1.56 f 0.27 1.48 0.42 1.50 f 0.09 1.68 t 0.30 1.57 f 0.50 1.29 ? 0.23

1.29 t 0.33 0.93 f 0.14 1.73 0.31 1.23 f 0.23 1.37 0.12 0.97 f 0.1 I

2.37 f 0.16 2.20 f 0.68 2.46 f 0.36 2.65 f 0.72 2.08 f 0.22 1.79 f 0.16"

1.89 -t 0.19 I .97 f 0.10 1.92 f 0.38 1.48 f 0.36 1.59 f 0.39 1.33 f 0.21

2.89 f 0. I9 2.35 tO.13" 2.32 f 0.25 2.68 f 0.51 2.40 f 0.09" 1.90 f 0 . 1 5 c

0.91 f 0.19 1.15 f0.32 1.27 f 0.16 0.98 f 0.07 1.42 f 0.17 1.69 2 0.19"

1.38 0.18 1.36 f 0.48 1.55 f 0.16 2.60 t 0.31 3.82 f 1.37 2.40 t 0.24'

1.02 f 0.19 0.96 f 0.19 1.05 f 0.21 0.97 t 0.17 0.99 2 0.08 0.92 f 0.05

I .95 t 0.27 1.61 f 0.46 2.07 f 0.28 3.75 f 0.72" 3.93 f 0.96 4.07 0.58"

2.50 f 0.37 2.28 t 0.29 2.73 f 0.43 2.69 t 0.29 2.52 f 0.30 2.60 f 0.35

2.1 I t 0.18 1.66 f 0.28 2.07 f 0.20 3.68 f 0.37' 3.08 f 0.06* 3.20 f 0.63

*

*

*

*

*

*

*

+_

*

*

*

* *

*

All guinea-pigs were killed by microwave irradiation (7 s) at fixed intervals after administration of soman (31.2 pdkg s.c.). Data are mean 2 SE values (in pmol/g wet weight of tissue).

A statistically significant difference from control values is indicated as follows: "p < 0.05, ' p < 0.001, ' p < 0.01.

J. Neumhem.. Vol. 54, NO.1. 1990

BRAIN TRANSMITTER CHANGES AFTER SOMAN were unchanged in all regions following soman administration. GABA levels showed a significant rise by 60 min in the cortex, hippocampus, and striatum with a lesser increase in the cerebellum and no change in the medulla-pons and midbrain-hypothalamus.

DISCUSSION This study gives the first extensive comparison of change in content of different transmitters after intoxication with the anticholinesterase soman. These changes occur both globally and in discrete brain regions with a time course that varied markedly for the different transmitter systems. The primary change following soman poisoning was a decrease in brain AChE activity and an elevation in ACh content at the onset of symptoms of anticholinesterase poisoning. The changes in levels of noncholinergic transmitters and their metabolites lagged behind those for ACh and Ch, a finding suggesting a secondary action in response to the elevated ACh levels. Significant increases in ACh content occurred in the cortex, striaturn, and hippocampus, which are the areas rich in cholinergic nerve terminals. There was no apparent correlation between the inhibition of AChE produced by soman administration and the elevation in ACh levels or between the elevation of ACh content and the control levels of neurotransmitter present. One interesting finding was that levels of ACh were significantly increased in the striatum when AChE was much less inhibited in this area than other areas showing no elevation of ACh level. This may be due to the higher control levels of AChE present in this area. The results obtained with guinea-pigs in this study parallel those reported in the rat after administration of soman (Shih, 1982; Lundy and Shih, 1983) and paraoxon (Wecker and Dettbarn, 1979). The results suggest that soman increases the steady-state concentration of ACh to a maximum that cannot be exceeded and that this leads to a feedback inhibition of ACh synthesis, which results in the level of ACh reaching a plateau with time. Also, ACh levels may have been prevented from further increase by compensatory feedback control by other transmitter systems, because many of the transmitters quantified in this study showed changes following soman poisoning. The brain content of Ch was elevated by 15-30 min in all areas except the cortex but declined back to control levels by 4 h. A similar time course of events was reported by Shih (1982) with soman and Rynn and Wecker (1986) with soman and sarin, but not DFP and paraoxon, in the rat. This Ch level increase is not characteristic of all organophosphate AChE inhibitors and appears to be unrelated to either AChE inhibition or elevated levels of ACh in brain. Increased levels of Ch and free fatty acids have been reported following ischaemia and administration of other convulsant drugs demonstrating enhanced hydrolysis of phosphatidylcholine and phosphoinositide. Pretreatment with

77

the anticonvulsant diazepam significantly attenuates the increase in Ch and free fatty acid levels induced by sarin and soman (Rynn and Wecker, 1986, 1987). This suggests that the effects of these agents on Ch metabolism are not due to excessive cholinergic activity but may be a consequence of the excitotoxic actions of these compounds. The earliest detectable change in level of a noncholinergic transmitter was a decrease in NA content. This most likely reflects an increased release of NA in response to the raised ACh level, because both oxotremorine, a muscarinic receptor agonist, and physostigmine have been shown to accelerate NA turnover in rat brain (Corrodi et al., 1967). Although the levels of DA itself were initially unchanged, the levels of its metabolites were markedly increased in DA-rich areas within the same time scale as the change in NA content. These findings are consistent with a n increased turnover of DA, with the DOPAC/DA ratio indicating a n increased DA utilisation (Hallman and Jonsson, 1984). Changes in the HVA/ DA ratio are similar to but follow those of DOPAC/ DA, a finding consistent with DOPAC being the chief metabolite and then HVA formed by 0-methylation of DOPAC (Dedek et al., 1979). At later time points, levels of DA itself were raised, and the changes in levels of metabolites were apparent in all brain regions, a result suggesting an increased synthesis in excess of that necessary to compensate for the increased release. 5-HT turnover, as measured by 5-HIAA/S-HT ratios, was increased after soman intoxication; these changes were weak compared with those in DA values and were localised to the cortex, hippocampus, and striatum. This correlates with the most marked changes in ACh levels occumng in these areas and is consistent with an interaction between cholinergic and 5-HT neurones. Similar results have been reported in the rat with physostigmine (Zarkowsky and Allimets, 1976) and with soman but not paraoxon (Prioux-Guyonneau et al., 1982) and in the rabbit with DFP (Koehn and Karczmar, 1978). Changes in levels of transmitter amino acids were restricted to the cortex, hippocampus, and striaturn, again correlating with those areas exhibiting a significant elevation of ACh levels. The observation that the GABA content is increased by soman administration in this study is supported by the findings of Ho et al. ( 1984). who found that toxic, but not nontoxic, doses of DFP increased levels of GABA and its precursor Glu and increased (6 h) and then decreased (24 h) GABA uptake and release. In contrast, no change in GABA levels was seen with nontoxic doses of soman (Coudray-Lucas et al., 1984; Liu et al.. 1988). The decreased content of the excitatory transmitter Asp and the increased content of the inhibitory transmitter GABA are consistent with a raised level of excitability. In rats. Wade et al. ( 1 987) could only show an increased release of Asp and Glu following soman administration when animals were exhibiting seizures. Similar changes J Neurochem , Lhl 54. No 1. 1990

P. FOSBRAEY ET AL.

78

in the content of transmitter amino acids have been demonstrated in other drug-induced seizure models (Chapman et al., 1985; Allen et al.. 1986). raising the question as to whether the transmitter changes result from soman intoxication or are secondary to seizure activity. However, Chapman et al. (1 984) have clearly demonstrated that transmitter changes produced by various convulsants precede any seizure activity, and Liu et al. (1988) were unable to find any relationship between the change in GABA. or ACh. content and the severity of seizures evoked by soman. The difficulty in detecting changes in levels of transmitter amino acids may reflect on using content as the method of assessment. Amino acids have complex metabolic as well as transmitter roles; as little as 20% ofthe content ofGlu may be involved in the transmitter function (Paulsen and Fonnum, 1988). Interpretation of the transient increase in Gln content is complicated by the involvement of Gln in both the synthesis and metabolism ofboth Glu and GABA (Paulsen and Fonnum, 1988) as well as other aspects of metabolism (Chapman et al., 1977). The changes in transmitter levels in this study show marked similarities to those produced in ischemic and hypoxic injury, where excessive release of DA is thought to potentiate the excitotoxic actions of Glu (Globus et al., 1988). These neurochemical findings are corroborated by the histological studies of neuronal damage in both ischaemia and soman intoxication (McLeod et al., 1984). Although ischaemia itself is unlikely to account for the early neurotransmitter level changes after soman administration, a contribution cannot be ruled out at later stages when animals exhibit respiratory difficulties. In conclusion, this study has clearly demonstrated by the concomitant measurement of neurotransmitter levels that changes occur in many ofthe major neuronal systems following the administration of soman to guinea-pigs. The change in ACh levels precedes that of any other neurotransmitter, a finding indicating this to be the primary change. The areas exhibiting changes in ACh content showed the greatest changes in noncholinergic transmitters, a result indicating their possible dependence on changes in the cholinergic system. This work has given clear indications as to the main transmitters and brain regions affected in soman intoxication, which will be further investigated by the use of more direct measurements of transmitter release. The study will also be extended to assess the effects of treatments for soman poisoning and other toxic agents. Acknowledgment:We would like to thank May Irwin and Suzanne Kinnear for their technical assistance.

REFERENCES Allen 1. C., Grieve A., and Griffiths R. (1986) Differential changes in the content of amino acid transmitters in discrete regions of the rat brain prior to the onset and during the course of homocysteine-induced seizures. J. Neurochem. 46, 1582- 1592.

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Barnes L., Karczmar A. G.. and Ingerson A. (1975) Effects of DFP on rabbit brain serotonin. Pharmacologist 17,80. Chapman A. G., Meldrum B. S.. and Siesjo B. K. (1977) Cerebral metabolic changes during prolonged epileptic seizures in rats. J. Neurochem. 28, 1025-1035. Chapman A. G., Westerberg E.. Premachandra M., and Meldrum B. S. (1984) Changes in regional neurotransmitter amino acid levels in rat brain during seizures induced by L-allylglycine. bicuculline, and kainic acid. J. Neurochfm. 43,62-70. Chapman A. G., Cheetham S. C., Hart G. P., Meldrum B. S.. and Westerberg E. (1985) Effects of two convulsant O-carboline derivatives. DMCM and 0-CCM. on regional neurotransmitter amino acid levels and on in vitro ~['H]aspartaterelease in rodents. J. Neurochem. 45,370-38 I. Corrodi H., Fuxe K., Hammer W., Sjcqvist F., and Ungerstedt U. (1967) Oxotremorine and central monoamine neurons. Lfe Sci. 6,2557-2566. Coudray-Lucas C., Prioux-Guyonneau M., Sentenac H., Cohen Y., and Wepieme J. (1984) Effects of physostigmine, paroxon and soman on brain GABA level and metabolism. Acta Pharmacol. To.yicol. (Copenh.)55, 153-157. Dedek J., Baumes R., Tien-Duc N., Gomeni R., and KorfJ. (1979) Turnover of free and conjugated (sulphonyloxy) dihydroxyphenylacetic acid in rat striatum. J. Neurochem. 33, 687-695. Ellman G. L., Courtney K. D., Andres V., and Featherstone R. M. (196 I ) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95. Fernando J. C. R., Hoskins B., and Ha I. K. (1986) The role of dopamine in behavioral supersensitivity to muscarinic antagonists following cholinesterase inhibition. Life Sci. 39,2 169-21 76. Flynn C. J. and Wecker L. (1986) Elevated choline levels in the brain. A non-cholinergic component of organophosphate toxicity. Biochem. Pharmacol. 35, 3 1 15-3 12 I. Flynn C. J. and Wecker L. ( 1 987) Concomitant increases in the levels of choline and free fatty acids in rat brain: evidence supporting the seizure-induced hydrolysis of phosphatidylcholine. J. Neurochem. 48, I 178- 1 184. Glisson S . N., Karczmar A. G.. and Barnes L. (1974) Effects of diisopropylphosphofluoridate on acetylcholine, cholinesterase and catecholamines of several brain parts of rabbit brain. Neuropharmacology 13,623-63 I . Globus M. Y.-T., Busto R., Dietrich W. D., Martinez E., Valdes I., and Ginsberg M. D. (1988) Effect of ischemia on the in viva release of striatal dopamine, glutamate, and y-aminobutyric acid studied by intracerebral microdialysis. J. Neurochem. 51, 14551464. Glowinski J. and lversen L. L. (1966) Regional studies of catecholamines in the rat brain. 1. The disposition of [3H]norepinephrine, [3H]dopamine, and ['HIDOPA in various regions of the brain. J. Neurochem. 13,655-669. Hallman H. and Jonsson G. ( I 984) Neurochemical studies on central dopamine neurons-regional characterization of dopamine turnover. Med. Biol. 62, 198-209. Ha I. K., Fernando J. C. R., Sivam S. P.. and Hoskins B. (1984) Roles of dopamine and GABA in neurotoxicity of organophosphorus cholinesterase inhibitors. Proc. W e s ~Pharmacol Soc. 27, 177-180. Hoskins B., Fernando J. C. R., Dulaney M. D., Lim D. K., Liu D. D., Watanabe H. K., and Ha 1. K. (1986) Relationship between the neurotoxicities of soman, sarin and tabun, and acetylcholinesterase inhibition. Toxicol. Left. 30, 121-129. Inns R. H. and Leadbeater L. (1983) The efficacy of bispyridinium derivatives in the treatment of Organophosphate poisoning in the guinea-pig. J. Pharm. Pharmacol. 35,427-433. Jovic R. C. (1973) Correlation between signs of toxicity and some biochemical changes in rats poisoned by soman. Eur. J. Pharmacol. 25, 159- 164. Koehn G. L. and Karczmar A. G. (1978) Effect of diisopropylphosphofluoridate on analgesia and motor bebaviour in the rat. Prog. Neuropsychopharmacol. 2, 169- 177. Liu D. D., Ueno E., Ha 1. K., and Hoskins B. (1988) Evidence that alterations in y-aminobutyric acid and acetylcholine in rat striata

BRAIN TRANSMITTER CHANGES AFTER SOMAN and cerebella are not related to soman-induced convulsions. J. Neurochem. 51, 181-187. Lundy P. M. and Shih T.-M. (1983) Examination ofthe role ofcentral cholinergic mechanisms in the therapeutic effects of HI-6 in organophosphate poisoning. J. Neurochem. 40, 132 1-1328. McLeod C. G., Singer A. W., and Hamngton D. C. (1984) Acute neuropathy in soman-poisoned rats. Neurotoxicology5, 53-58. Paulsen R. E. and Fonnum F. (1988) Regulation of transmitter yaminobutyric acid (GABA) synthesis and metabolism illustrated by the effect of y-vinyl GABA and hypoglycemia. J. Neurochem. 50, 1151-1 157. Potter P. E., Hadjiconstantinou M., Rubenstein J. S., and Neff N. H. ( 1985) Chronic treatment with diisopropylfluorophosphate increases dopamine turnover in the stnatum of the rat. Eur. J. Pharmacol. 106,607-61 1. Prioux-Guyonneau M., Coudray-Lucas C., Coq H. M., Cohen Y., and Wepierre J. (1982) Modification of rat brain 5-hydroxytryptamine metabolism by sublethal doses of organophosphate agents. Acta Pharmacol. Toxicol. (Copenh.) 51, 278-284.

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Shih T.-M. (1982) Time course effects of soman on acetylcholine and choline levels in six discrete areas of the rat brain. Psychopharmacology (Berlin) 18, 170- 175. Wade J. V., Samson F. E., Nelson S. R., and Pazdemik T. L. (1987) Changes in extracellular amino acids during soman- and kainic acid-induced seizures. J. Neurochem. 49,645-650. Wecker L. and Dettbarn W. D. (1 979) Relationship between choline availability and acetylcholinesynthesis in discrete regions of rat brain. J. Neurochem. 32,96 1-967. Wecker L., Mobley P. L., and Dettbarn W. D. (1977) Central cholinergic mechanisms underlying adaptation to reduced cholinesterase activity. Biochem. Pharmacol. 26,633-637. Wetherell J. R., Fosbraey P., and French M. C. (1989) A comparison of the distribution of neurotransmitters in brain regions of the rat and guinea-pig using a chemiluminescentmethod and HPLC with electrochemical detection. J. Neurochem. 53, 15 19- 1526. Zarkowsky A. M. and Allimets L. H. (1976) Influence exerted by cholinergic drugs on the serotonin metabolism and emotional reactions of rats. Farmakol. Toksikol. 39, 26 1-264.

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