Act0 pharmacol. et toxicol. 1978, 43, 169-173.

From the Brain Research Laboratory, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh-202001, India.

Levels of Dopamine, Norepinephrine and 5- Hydroxytryptamine in Different Regions of the Rat Brain in

Thallium Toxicosis

BY Mobdi Hasan, S. Fatehyab AU and M. Tariq*

(Received August 9, 1978; Accepted February 27. 1978)

Abstract: The levels of dopamine. noradrenaline and 5-hydroxytryptamine were estimated in the hypothalamus and limbic areas, corpus striatum, cerebellum and the brain-stem of rats administered thallous acetate (5 mgkg i.p.) daily for 7 days. Significant decreases in the concentration of dopamine occurred in the hypothalamus, limbic areas and corpus striatum. Noradrenaline did not reveal signific- ant alteration in any of the regions of the brain examined. The concentration of 5-hydroxytryptamine was significantly lowered in the corpus striatum. cerebellum and the brain-stem.

Key-words: Thallium acetate - dopamine -norepinephrine - 5-hydroxytryptamine - regional levels - rat brain.

The initial symptoms of thallium intoxication are frequently neurological and include dyses- thesia, neuropathy, chorea, athetosis, excite- ment, sleeplessness, tremors, convulsions, mental aberrations, hysterical laughter, motor weakness, nerve palsies and coma (Bank et al. 1972; Reed et al. 1963). Although this heavy metal is a cumulative poison of high toxicity, its pathophysiology is not yet clearly understood (Cavanagh et al. 1974). Starke et al. (1969) showed that catecholamine metabolism was normal in rats given thallium; however the ATP

* Postgraduate Research Laboratory, A.K. Tibbiya College, A.M.U. Aligarh.

ases of aminestoring granules from bovine ad- renal medulla were inhibited by Tl*** (Burger & Starke, 1969). If T1* was oxidized to TI*** in the organism, th is inhibition could be re- sponsible for altered catecholamine metabolism in thallium intoxication, but this has not yet been demonstrated in man (Bank et al. 1972). Interestingly, treatment of choreiform sequelae of thallitoxicosis by levodopa has recently been described by Patterson (1975). It would, there- fore, be of interest to investigate the effect of thallium intoxication on the levels of dopamine, noradrenaline and 5-hydroxytryptamine in the hypothalamus and limbic areas, corpus striatum, brain stem and cerebellum of the rat.

170 M. HASAN, S. FATEHYAB ALI AND M. TARIQ

Materials and Methods Animals: Male albino rats (Charles Foster Strain) weighing 150-200 g were used. The animals were fed pellet diet (Hindustan Lever Ltd., India) and water ad libitum. They were divided into two groups of 15 (Group 1 - experimental) and 10 (Group 2 - control) respectively.

Thallium Administration: Thallous acetate and other chemicals of analar grade were obtained from B.D.H. (England). Thallous acetate, dissolved in distilled water, was injected intraperitoneally daily for one week in the rats (5 mgkg) of experimental group. The total dose administered was 35 mg thal- liumkg, corresponding to the LDso dose of Brewer & Haggerty (1958). The control group was treated in a similar manner with equal volumes of sodium acetate solution of the same molar concentration.

Removal ofBrain: The rats were sacrificed by de- capitation, the brains were removed within 15-20 sec. and their hypothalamus and limbic areas (Gyrus cinguli, hippocampus and orbito-temporo-insular cortex), corpus striatum, brain stem and cerebellum were rapidly dissected out in a cold room at a temper- ature of 14+2”. They were weighed to the nearest milligram on a single pan electrical balance (1OO-5OO mg) and separately homogenized. Glass stoppered bottles sized 30, 60 and 125 ml were used in the solvent extraction procedure.

Extraction: Extraction of all these compounds was done by the method of Welch & Welch (1969) except that the time for shaking at each solvent extraction was increased to 20 min. The temperature throughout these operations ranged between 10” and 15”.

All the reagents used were of analytical grade. Stock solutions of dopamine, noradrenaline and 5- HT were prepared in 0.01 N-HC1 and stored in the deep freeze. They were finally diluted to give a con- centration of 1 pg/ml. A 20 per cent homogenate of the different regions of the brain was prepared in cold 0.01 N-HCl to which 0.1 ml of 10% EDTA was added. Three millilitre of homogenate in each case was added to 25 ml n-butanol in a 60 ml glass stop- pered bottle containing 4 g NaCl. The bottles were shaken for 20 min. on a reciprocating shaker at about 250 excursions per min. and centrifuged at 3000 rpm for 8 min. in the cold. The butanol (24 ml) was decanted into a 125 ml glass-stoppered bottle con- taining 40 ml n-heptane; 1.5 ml of 0.5 M pH 7.3 phosphate buffer. The bottles were shaken again for 20 min. and centrifuged at 2000 rpm for 8 min. in the cold. At this stage, it was important that the pH of the phosphate buffer was not allowed to drop below 7.0. Phosphate buffer (1.5 ml), which increased slightly in volume due to water driven from the butanol phase after the addition of heptane, was transferred to a clean 30 ml bottle and acidified with 3 N-HCl to pH 3.5-4.0. Thereafter, 20 ml of peroxide-free ether was

added and the bottles were shaken for 20 min. and centrifuged in the cold. Three 0.5 ml aliquots of the acid-aqueous layer were taken directly from the bot- tom of the ether extraction bottles with a 0.5 ml vol- umetric pipette and were refrigerated for the estima- tion of NA, DA and 5-HT. 5-hydroxytryptamine was estimated on the same day, whereas dopamine and noradrenaline were estimated on the following day. Recovery experiments were performed taking the known standards of the compounds in 3.0 ml of 0.01 N-HCl and then carrying out all the earlier men- tioned steps.

Fluorometric analysis. All these compounds were analysed using the method of Welch & Welch (1969). Fluorescence was measured in a Turner Mod- el 430 Spectrophotofluorometer (G.K. Turner As- sociation U.S.A.).

IHydroxytryptamine. - To each of the 0.5 ml samples, obtained in the extraction procedure, was added 1.5 ml of 6-N-HCl. One sample was acidified at a time and its fluorescence was read at 295/535 nm promptly.

Norepinephrine. - To the 0.5 ml samples of the extract collected in small test tubes, the following reagents were added in order: 0.5 ml (2 M, pH 6.8) acetate buffer; 0.1 m l O . 1 N iodine solution; 0.15 ml 0.1 N sodium thiosulphate and 0.2 ml alkaline ascor- bic acidlethylene diamine solution. The test tube rack was placed under a fluorescent desk lamp with the tops of the tubes positioned about 5-10 mm. Pluorescence was read at 400/510 nm within 35 min.

Dopamine. - To the 0.5 ml sample, the following reagents were added, at 5 min. intervals: 0.5 ml(2M, pH 6.8) acetate buffer; 0.1 mlO.1 N iodine solution; 0.2 ml alkaline sodium sulphite/EDTA solution and 0.25 ml 1 : 1 glacial acetic acidkoncentrated HC1. All the test tubes were placed in a boiling water bath for 45 min., after which they were allowed to cool at room temperature before reading. The fluorescence was read at 335/380 nm within one hr. after develop- ment.

Statistical analysis. The data were analysed statis- tically using Student’s “t” test and significant differ- ences between the means of the treated and the con- trol groups and the “p” values calculated.

Results Following the administration of thallous acetate to rats, signs such as irritability, ataxia and at times convulsions were observed. After 5 days the rats usually became lethargic and the motor activity was considerably diminished. Five rats belonging to group 1 died during the experi- ment. Table 1 shows recoveries of dopamine, noradrenaline and 5-hydroxytryptamine. The

CATECHOLAMINES IN THALLITOXICOSIS 171

Table 1

Representative recoveries of dopamine, norepinephrine and 5-HT

Compound Amount taken Amount recovered % Recovery )rg w

Dopamine Norepinephrine 5-HT

0.2 0.1 0.2

0.144% 0.003 0.085'+ 0.007 0.156'2 0.008

72 85 78

a Mean f S.E.is based upon 10 samples

concentrations of dopamine, norepinephrine and 5-HT in experimental thallium toxicity are given in Table 2. Significant reduction in the levels of dopamine was observed in the hypothalamus, limbic areas (P < 0.05) and the corpus striatum (P< 0.01). Noradrenaline con- centration showed no significant alteration in any of the regions investigated in the present study. But the levels of 5-HT showed signific- ant diminution in the corpus striatum (p < 0.001), brain-stem (P < 0.001) and cerebellum (P < 0.05).

Discussion The administration of thallous acetate (5 m e ) for one week to rats resulted in a significant fall in the levels of dopamine in the hypothalamus, limbic areas and corpus striatum; and that of the 5-HT in the corpus striatum, brain-stem and cerebellum. The concentration of noradrenaline did not alter in thallium-intoxicated tats as com- pared to controls. Regional heterogeneity was apparent in the thallium-induced reduction of dopamine and 5-HT levels. The present investi- gation was carried out in four different regions of the brain to reveal regional characteristics of thallium toxicity. It is of interest that Freeman & Giarman (1962) and Schanberg C Giarman (1962) have emphasized the point that these levels reflected the net result of a number of component processes. Changes in the level of brain dopamine are known to be associated with certain motor dysfunctions (Hornykiewicz 1966). Moreover Freed et al. (1976) have

shown a marked lowering of the dopamine level of the corpus striatum after Mipafax toxicosis. The fall in the dopamine concentration of cor- pus striatum observed in this study is of particu- lar interest in view of the beneficial effect of levodopa reported by Patterson (1975) in cases of choreiform sequelae of thallitoxicosis. On the other hand, Buschke & Peiser (1922) have highlighted the endocrine and autonomic dis- turbances caused by thallium. Moreover Prick et al. (1955) described mesodiencephalic dis- turbances due to thallium intoxication. They ex- plained the occurrence of sleep disturbances, diabetes insipidus and Korsakow's symptom complex observed in cases of thallium poison- ing as being due to the involvement of auton- omic zones. The lowering of dopamine levels in the hypothalamus and limbic areas thus substan- tiates the reported clinical observations. A con- siderable body of anatomical, pharmacological and neurophysiological evidence links central serotonergic mechanisms to the regulation of such diverse functions as sleep and body temp- erature, as well as to the pathophysiology of human disturbance ranging from schizophrenia to Parkinsonism (Chase 1974). It is of interest to note that significant lowering of 5- hydroxytryptamine concentrations occurred in the corpus striatum, brain-stem and cerebellum. From our investigations it may, therefore, be suggested that there is a significant disturbance in the turn-over of dopamine and 5- hydroxytryptamine in different regions of the rat brain, the mechanism of which requires further investigations.

Tab

le 2

Lev

els

of d

opam

ine,

nor

epin

ephr

ine

and

5-H

T in

diff

eren

t reg

ions

of

the brain

(exp

ress

ed as

pg

/g f

resh

wei

ght

Mean f S.E.) of

thallium

toxi

cosi

s (5

mg&

g i.p

. da

ily f

or 7

day

s)

K co

mpo

und

Hyp

otha

lam

us

Perc

ent

Cor

pus

stria

tum

Pe

rcen

t C

ereb

ellu

m

Perc

ent

Bra

in s

tem

Pe

rcen

t and

limbi

c ar

ea

chan

ge

chan

ge

chan

ge

chan

ge

g C

ontro

l Experi-

Con

trol

Experi-

Con

trol

Experi-

Con

trol

Experi-

men

tal

men

tal

men

tal

men

tal

?

N=

10

N=

10

N=

10

N=

10

N=

10

N=

10

N=

10

N=

lO

v

Dop

amin

e 0.

360

0.18

0*

-50

0.89

7 0.

328*

* -64

0.406

0.27

3 -3

3 0.

515

0.38

7 - 2

5 f

- + f

f

f

f

f

f

0.06

0.

03

0.16

0 0.

08

0.05

0.

03

0.11

0.

09

Nor

epin

e-

0.85

4 0.

783

-9

2.18

4 1.

761

-20

1.01

3 0.

681

-33

1.27

2 0.

883

-31

phrin

e f

f

f

f

f

f

-c f

0.24

0.19

0.

59

0.24

0.

26

0.17

0.

41

0.27

5-H

T

0.200

0.13

2 -3

4 0.

555

0.26

4***

-5

3 0.

382

0.24

5*

-36

0.66

3 0.

226*

**

-66

2

f

f

f

- -1- -c

2

f

0.04

0.03

0.

02

0.02

0.

03

0.04

0.04

0.03

x

Eac

h va

lue

is th

e m

ean of o

bser

vatio

ns

* P

L0.

05;

** P

LO

.01;

**

* PL

O.0

01

CATECHOLAMINES IN THALLITOXICOSIS 173

A c k n o w l e d g e m e n t s

Thanks are due to the C.S.I.R. (India) for awarding a Research Fellowship to one of us (S.F.A.) and to Mr. G. Madar for technical as- sistance.

References Bank, W.J., D.E. Pleasure, K. Suuki, M. Nigro &

R. Katz: Thallium poisoning. Arch. Neurol. 1972, 26, 456-464.

Brewer, E. & R.J. Haggerty: Toxic Hazards. Rat poison. III Thallium, Strychnin and ANTU. New. Engl. J. Med. 1958, 259, 1038-1040.

Burger, A. & K. Starke: Beeinflussing der ATP ase aminspeichernder Granula von Nebennierenmark und Milznerven durch Thallium. Experientia.

Buschke, A. & B. Peiser: Die Wirkung des Thallium auf das endokrine System. Klin Wchensch. 1922, 2 , 995.

Cavanagh, J.B., N.H. Fuller, H.R.M., Johnson& F. Rudge: The effect of thallium salt with particular reference to nervous system changes. A report of three cases. Quart. J. Med. 1974, 431170, 293- 319.

Chase, T.N. : “Serotonergic mechanisms and ex- trapyramidal function in man”. Advances in Neurology, (Mc Dowell, F.H. and Barbeau, A. eds) Vol. 5, Raven Press, New York, 1974, pp.

1969, 25, 578-579.

31-39.

Freed, V.H.. M.A. Matin, S.C. Fang & P.P. Kar: Role of striatal dopamine in delayed neurotoxic effects of organophosphorus compounds. Euro- pean J. Pharmacol. 1976, 35, 229-232.

Freeman, D.X. & N.J. Giarman: LSD-25 and the status and level of brain serotonin. Ann. N.Y. Acad. Sci. 1962. 96, 98-107.

Hornkiewicz, 0: Dopamine (3-hydroxytyramine) and brain function. Pharmncol. Rev. 1966, 18,

Patterson, J.F.: Chronic thallitoxicosis. Treatment of the choreifom sequelae. South Med. J. 1975.68,

Prick, J.J.G., W.G.S. Smith & L. Muller: Thallium poisoning. Elsevier Publishing Co. Amsterdam, London, New York, 1955, pp. 55.

Reed, D., J. Crawley, S.N. Faro, S.J. Pieper & L.T. Kurland: Thallitoxicosis. Acute manifestation and sequelae. J. Amer. Med. Ass. 1963, 183, 516- 522.

Schanberg, S.M. & N.J. Giarman: Drug-induced alt- erations in the sub-cellular distribution of 5- hydroxytryptamine in rat brain. Biochem. Phar- macol. 1962, 11, 187-194.

Starke, K., A. Burger, & H.J. Schumann: Thallium and brenzcatecholamin-Stoffwechsel* Thallium and Catecholamine metabolism. Naunym Schmiedberg Arch. Pharm. 1969, 264, 310-31 1 .

Welch, A.S. & B.L. Welch: Solvent extraction for simultaneous determination of norepinephrine, dopamine, serotonin and 5-hydroxy indol acetic acid in a single mouse brain. Analyt. Biochem.

925-929.

923-925.

1969, 30, 161-169.