Comparison of the chronic effects of α-chlordane and pp′ DDT on the level of cerebral amino acids and pree ammonia in mice

Pharrnaco/og/ca/ Research Commun/cat/ons, Voi. 9, No. 7, 1977

613

COMPARISON OF TH~ CHRONIC EFFECTS OF c~-CHLORDANE AND ppt DDT ON THE LEVEL OF CEREBRAL AMINO ACIDS AND FRES A~.ONIA IN MICE

M.A.Matin, P.P.Kar, M.Z.Hasan and M.Anand Industrial Toxixology Research Centre, Lucknow, U.P., India

R~ei~d180cmber1976 SUMMARY

Treatment with pp'DDT (25 mg/kg/day) orally for 45 days

produced ataxia in mice and a reduction in the level of cerebral taurlne, glycine and GABA. The concentration of cerebral free ammonia was not altered in pp' DDT treated animals. Treatment with the same dose of

@~Chlordane for the same period neither produced ataxia in

animals nor any change.in the level of cerebral amino acids or free ammonia. Thus ~-chlordane lacks the chronic effects of pp' DDT on central nervous system.

I~TRODUCTION

The c h l o r i n a t e d

hydrocarbon insecticides

produce t o x i c

signs- hyperexcitability, tremors and convulsions- which are relate~ to the concentration of the compound in the brain (Dale et al, 1963; Woolley and Barton, 1968). Changes in the level of brain ammonia (Hathway and Mallinson, 1964; Hathway et ai,1965), acetylcholine (Omer and Ecobichon, 1971) and other biogenic amines (Peters et ai,1971; Hrdina et ai,1971) have been reported following the acute administration of these compounds. It has recently been suggested that amino acids act as transmitters at certain central synapses (Phillis, 1970). Changes in the level of aminoacids have been reported to modify the behavioural pattern or excitability of the brain (woodbury and Yernadakis, 1950; Woodbury and Esplin, 1959; Hayashi and Nagai, 1956). Since it is the chronic exposure to small doses of chlorinated hydrocarbon insecticides which actually presents

Pharmacological Research Communications, I/o4 9, No. 7, 1977

614

a ~otential health hazard, we examined the lone term effectof ~-Chlordane and pp' DDT on the level of cerebral amino acids

and free ammonia in

mice.

METHODS

Adult male albino mice~ 20 +_.5 g., were used. The animals were

divided into three groups and each group was subdivided into subgroups of three mice. From these subEroups~ the cerebral hemisgheres were seperately pooled for the ~etermination of amfno acids or free ammonia concentration.

c~-chlordane or pp' DDT were dissolved in peanut oil and given

orally to animals. The animals of group one served as controls and wet given the oily vehicle._The animals-of gro~p.tworand ~hree received ~-chlordane

or DP' DDT (each 25 mg/kg) daily f o r 4 5

davs. The~aniMa~s

were kept in individual cages a n ~ w e r e given food and water ad libi~um~ They were observed carefully for occurrence of any apparent neurotoxio symptoms or changes in gross behaviour. The animals were killed by immersion inliquid air 24 hr. after the last treatment. The cerebral cortex was quickly dissected from the frozGn brain stem and was homogenized in 12~ ice cold trichlor _~ce\ticacid and centrifuge~. Free ammonia.in th,,supernatant~was determined b y % h e

method of Conway~(1947) as described by-Richter ahd

Dawson (1948) using a diffusion time of %0 minutes. Free,bound and total GABA level of the cer~braiCcortex was determined according to the method of Lovell?Ja/4dEliiott~6196~): as modified by Tabia et al (1961)~.~0%heramino acids were determined by the method of Awapara (1948) The data were analysed sta~is%Ically-using ~heStuden%s %test and significant differences between the means de%ermined.

Pharmacological Research Communications, VoL 9, No. 7, 1977 RESULTS

615

Treatment with C~-chlordane or pp' DDT did not produce

any signifioan~ change in the concentration of cerebral free @mmonia in mice (Table I). TABLE i. Effect o f ~-chlordane and pp' DDT treatment on the concentration of cerebral free ammonia in mice. Each figure represents the mean i SEM of 6 groups of animals (each group consisting of subgroups of 3 animals).

F r e e ammonia(~ Mole/g)

Treatment

-mean.+ SEN

p value

"

Controls

0.20 + 0.01

N.S.

@~-chlordane~ 25 mg/kg/day for 45 d a y s

0.17 + 0.02

N.S.

pp'DDT, 25 mg/kg/day for 45 days

0.22 + 0.02

N.S.

N'S..

Not-si@ificant-

The effect of oC-chlordane and pp' DDT treatment on the level of variuus amino acids and GABA is given in Tables 2 and 3 respectively. Treatment with pp' DDT reduced the level of taurine and glycine (Table 2), free, bound and total GABA (Table 3). level of aspartic acid,

The

glutamic acid, glutamine and ~-alanine was

not significantly changed by pp' DDT treatment (Table 2). Treatment withE-chlordane 2 ana

did not change the level of cerebral amino acids

3). pp' DDT treated animals developed ~taxia during the lest

3-4 days of tae experimental period.

~-chlordane

treated animals

werenormal. No other changes i n gross behaviour were observed animals trested

DISCUSSION

,~ith either G-chlordane or pp' DDT.

It was previously reported that high or convulsive

of chlorinated

in

hydrocarbon insecticides

doses

increased the c o n c e n t r a t i o n

of

Pharmacologica/ Research Communicationsj Vol. 9, No. 7, 1977

616

Changes ~n t h e l e v e l o f c e r e b r a l amino a c i d s o f mice t r e a t e d with ~'chlordane or' pp' DDT (e~ch 25 mg/kg.,orally) daily for 4~ days. The values are expressed in ~ .~ble/g. Each figure represents the mean ~ SEMIof'6 grou'p~ of animals (each group consisting o f subgrouPs, of 3 animals).

TABLE 2

Aspartic acid

~-alanine

Olutamic acid

O1Fcine

Control

4.20+.0.29

0.56+0.04

7.60+0.12

3.02+_0o12 2.14+0.10

<-0h].ordane

4.1~+0.26

0.57+0.09

7.92_+0.49 3.23.+0.16 2.07,+0.18

pp DDT ' *

Olutamine

Taurlne

2.4~o.3l 2.32_+0.21

4°30_+0°23 0.54+0.04 7.t~3+0.24 2.42f.0.22 2.09+O.26 1.t~3+0o14 S i g n i f t o a n ~ l y different from values in ;ontroi animalsj~I P(.05)

cerebral free ammonia

which has stimulatory effects on central nervous

s~stem resulting in t~emors and convulsions (Hathway and Ma~linson,1964; Hathway etal,

1965~. According to our resuits, the concentration

of

cerebral free ammonia in animals treated daily with small doses (25 mg/kg) of Dp' DOT or o~-chlordane was not significantly different from the values in control animals (Table I). Further only pp' DOT treated animals developed ataxia while

oC-chlordane treated animals ~ere normal.

This indicates that changes in the concentration of cerebral ammonia, per se, are not respons;ible for araxla or delayed toxic effects of chlorinated hydrocarbon insecticides. The r e s u l t ~ of taurine,

glycine

indicate

significant

pp" U~'A° t r e a t m e n t

on c e n t r a l

n e u r o n s ( C u r t i s and H a t k i n s , 1 9 5 5 ) .

which ~id not produce a t a x i a effect

These f i n d i n g s ,

reduced the level

(TabAe 2) and GABA ( T a b l e 31) w h i c h h a v e b e e n r e p o r t e d

to exert a depressan% effect ~-chlordane

that

i n a n i m a l s , was v o i d o f

on t h e l e v e l o f c e r e b r a l

however, are not suggestive

any

amino a c i d s ( T a b l e s 2 and 3 ) . of a positive

relationship

between t h e development of a t a x i a and t h e lowering of c e r e b r a l

amino a c i d s .

Pharmacological Resesrch Communications, Vo4 9, No. 7. 1977 TABLE 3.

7

Oerebral GABA (free, bound and total) level in mice treated with pp' ~DT or ~4-chlordane ~each 25 mg/kg.,orally) daily for 45 d~ys. The values are expressed in ~ Mole/g° Each figure represents the mean ~ SEM of 6 groups o£ animals (each group consisting of subgroups of 3 animals).

Treatment

Total GABA

Free GABA

Bound GABA

Control

2.94±0.19

1.78t0.i0

1.27+..0.06

~-chlordane

2.8bt0.26

1.84±0.18

1.13t0.17

p ,DDT

2.3l+0 17

0.89 O.08 b

Significantly different from values in control animals (p <'05). b significantly

different

from v a l u e s i n c o h t r o l a n i m a l s (p < . 0 1 ) .

It was previously reported that the daily administration of pp' DDT ( 2 m~/100 E) to rats for four weeks produced stimulation or activa~io~ ~increase in frequency and amplitude) o£ EE~ (Desi et a1,1966; Farkas et al,

196~). I t wa~ a l s o r e p o r ¢ e d t h a t t h e r e l e a s e

of taurine

from t h e

c e r e b r a l c o r t e x was g r e a t e r d u r i n g a r o u s a l t h a n d u r i n g s l e e p ( J a s p e r and Koyama,1969). F u r t h e r t h e t a u r i n e c o n t e n t o f cerebrum o f a n i m a l s showing a synchronized

ESG p a t t e r n was h i g h e r t h a n i n t h e cerebrum o f a n i m a l s

showing an a c t i v a t e a level of taurine

pattern

~Guidotti et at,

i n pp'DDT t r e a t e d

1972)o Thus t h e r e d u c e d

a n i m a l s may be due t o DDT i n d u c e d

s t i m u l a t o r y e f f e c t s , on EEG. The r e d u c e d l e v e l of c e r e b r a l aABA i n pp'DDT t r e a t e d a n i m a l s ( T a b l e 3) i s a l s o i m p o r t a n t s i n c e t h e d e c r e a s e i n b r a i n GABA h a s p r e v i o u s l y been h e l d r e s p o n s i b l e f o r c e r t a i n

drug induced convulsions

( K t l l a m and B a i n , 1 9 5 7 ) and i n c r e a s e d b r a i n e x c i t a b i l i t y

(Woodbury and

V e r n a d a k i s , 195~). G l y c l n e , a n o t h e r amino a c i d r e d u c e d by pp'DDT t r e a t m e n t ( T a b l e 2~

(Johnson et aI,1970~.

also exerts inhibitory

effect

on c e n t r a l

neurons

Pharmacological Research Communications, Vol. 9, No. 7, 1977

618

It has been suggested that the balance between the excitatory ~nd inhibitory systems of the brain regulates the neuronal activity and determines the behavioural pattern (Roberts et al, 1958). It is possible that the reduction in the level of cerebral amino acids (Tables 2 and 3) which exert an inhibitory effect on central neurons (Curtis and Watkins, 1965), may disturb this balance resulting in motor •

1

dysfunction or ataxia in pp' DDT treated anlmals.

It may be concluded from our results that chronic treatment with ~Q-chlordane failed %0 produce ataxia or changes in the level or' amino acids which were observed after daily treatment with the same dose of pp' DDT.

Acknowledgement :

The authors are grateful to CIBA and VELSICOL for

the generous gifts of pp' DDT and

o~-chlordane respectively.

REFERENCES Awapara, J., (1948)

Arch. Biochem. 19

Conway,E.J.

Microdiffusion analysis and volumetric error, 2nd.ed., Crosby Lockwood, London, p. 87.

(1947)

, 172

Curtis,D.R and Watkins, J.C (1965) Pharmacol. Rev., 17 , 347

Dale,H.B., Gaines,T.B., Hayes,Jr.W.J. and Pearce,G.W (1963) Science, 142, 1474 Desi,I., Farkas,I and Kemeny,T (1966) Aota physiol. Aoad. Sci. Hung.,

30, 275 Farkas,I., Desi,I., and Kemeny,T (1968)

Toxicol. Appl. Pharmacol.,

I_2 , 518 Guidotti,A., Badiani,G and Pepeu,G (1972) J.Neurochem. 19 , 431

Hathway, DoE and Mallinson,A (1964)

Biochem. J. 90 ,51

Pharmacological Research Communications, VoL 9, No. 7, 1977

619

Hathway,D.E., Mallinson,A and AMintonwa, D.A.A (1965) Bioohem.J. 94, 676 Hayashi,T and Nagai,K (1956) Abstr.XX Intern.physiol.Congr.p. Hrdina, P.D., Singhal,R.L., Peters,D.A.~ and Ling,G.M (1971) J. Pharmaco]o I.~ , 379

410 Eur.

Jasoer,R.H and Koyama,r (1969) aan.a.Physiol. Pharmaool. ~4~ , 889 Johnson,E.S., Roberts,M~H.T and Straughan,D.W (1970) Brit.J.Pharmacol. 38, 659 Killam,K.F and Bain, J.A (1957) Y.pharmacol.exp.Ther. ~

, 225

Lovell,R.A and Elliott,K.A.C (1903) J.Neurochem. I_.O_, 479 0mer,V.V.St., and Eoobichon,D.J

1971) Can.J.physiol.Pharmacol.49,79

Peters,D.A.V., Hrdina,P.D., Sin~nai,R.L ana Ling,G.M (1971) Pharmacologist. 13 , 241

Phillis,W,J (A970) The pharmacolegy of synapses (Pergamon press, Lona) p. 223 Richter,D and Dawson,R.M.C (1948) J.bio].Chem.

I_~ , 1199

Roberts,E., Rothstein~M and Baxter~C.F (1958) Proc.Soc.expl.Biol.Med. 97, 796 Tabia,R., Pasantes,H., DeLa Mora,M.P., Or%ega,B.G and Massieu~G.H (1967) Biochem.pharmacol. 16 ~ 483 Woodbury,D.M and Vernadakis~A (1958) Fed.Proc.fed.Am.Soc.exp.Biol.17,420 Woodbury,D.M and Esplin,D.W (1959) neurochemistry of anticonvulsant dru~(Ed.Braceland,F), Baltimore:Williams & Wilkins,p.243 Woolley,D.E and Barron,B.A(196G) Toxicol.Appl.Pharmacol. 12 , 440