Bull. Environ. Contam. Toxicol. (1992) 49:402-409 9 1992 Springer-Verlag New York Inc.
i
Snvironmental Contamination and Toxicology
Differential Responses of Regional Brain Polyamines Following In Utero Exposure to Synthetic Pyrethroid Insecticides: A Preliminary Report Raghib Husain, Madhu Malaviya, Prahlad K. Seth, and Raushan Husain Industrial Toxicology Research Centre, Post Box 80, Mahatma Gandhi Marg, Lucknow-226 001, India Synthetic p y r e t h r o i d s are p o t e n t i n s e c t i c i d e s of high bloefficacy. T h e y are now b e i n g e x t e n s i v e l y u s e d for pest management in a g r i c u l t u r a l p r a c t i c e s in India. M a m m a l i a n c e n t r a l n e r v o u s s y s t e m is the p r i m a r y target for the toxic a c t i o n of the synthetic pyrethroids, which are c l a s s i f i e d into two c a t e g o r i e s a c c o r d i n g to the s i g n s and s y m p t o m s of t o x i c i t y e x h i b i t e d i.e. TypeI (T-Syndrome) and Type-ll (CS-Syndrome) (Aldridge 1990; V i j v e r b e r g and B e r c k e n 1990). The T-syndrome is characterlsed by hyperexcltation, tremor and c o n v u l s i o n s w h i l e C S - s y n d r o m e by an i n i t i a l h y p e r a c t i v e behavlour, facial tremor, profuse salivation and spontaneous choreathetosis. It is now w e l l e s t a b l i s h e d that pyrethroids exert their neurotoxic effects primarily by m o d i f y i n g s o d i u m g a t i n g k i n e t i c s and the neurotoxic p o t e n t i a l of T y p e - l l is c h i e f l y due to the o~-cyano-3-phenoxybenzyl alcohol moiety (Verschoyle and Aldridge 1980). Our e a r l i e r s t u d i e s w i t h fenvalerate g i v e n o r a l l y to adult rats s h o w e d m a r k e d i n h i b i t i o n in regional brain levels of catecholamines and their m e t a b o l i t e s ( H u s a i n et al. 1991). The e x t e n s i v e and i n j u d i c i o u s use of i n s e c t i c i d e s poses d i r e c t or i n d i r e c t h e a l t h h a z a r d , to man and live s t o c k as well as leads to environmental contamination. E x p o s u r e of p r e g n a n t w o m e n at the m a n u f a c t u r i n g sites, in the f i e l d s or t h r o u g h environmental contamination poses h e a l t h risk to d e v e l o p i n g h u m a n fetus. Insplte of numerous s t u d i e s c o n f i r m i n g that pyrethroids are n e u r o t o x i c to the adult n e r v o u s s y s t e m , l i t t l e is k n o w n about their perinatal effects. The fetal brain is e s p e c i a l l y v u l n e r a b l e to c h e m i c a l s w h i c h r e a d i l y cross the p l a c e n t a l b a r r i e r d u r i n g b r a i n s p u r t p e r i o d . Since Send above
reprint address.
requests
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during this time c e l l u l a r d e v e l o p m e n t and ontogenesis of various neurotransmitter system(s) progresses r a p i d l y and o f t e n the e f f e c t s i n d u c e d by the chemicals or drugs lead to f u n c t i o n a l and behavioural deficits w h i c h a p p e a r m u c h later in c h i l d h o o d . The p r e s e n t s t u d y was t h e r e f o r e a i m e d to investigate the in utero e f f e c t s of three t y p e - l l p y r e t h r o l d s vlz. Fenvalerate (Fv), C y p e r m e t h r l n (Cyp) and Deltamethrln (Dt) on the early brain development. Levels of polyamines reported to be closely associated with g r o w t h and d i f f e r e n t i a t i o n (Tabor and Tabor 1983) and the r e g u l a t i o n of DNA, RNA and p r o t e i n synthesls in many g r o w i n g systems ( B a c h r a c h 1973), w e r e s t u d i e d in s p e c l f [ c r e g i o n s of rat brain. MATERIALS
AND
METHODS
Putresclne dihydrochloride, spermidine trihydrochloride, spermlne tetrahydrochlorlde and d a n s y l chloride were obtained from Sigma C h e m i c a l s Co., St. Louis, USA. E m u l s i f i a b l e c o n c e n t r a t e s of f e n v a l e r a t e , deltamethrin and c y p e r m e t h r l n in 2 0 % , 2 . 8 % and 25% EC formulations, w/w r e s p e c t i v e l y were o b t a i n e d from Shroff Industrial Chemicals, Gujarat, India. All other reagents and solvents used w e r e of the highest analytical grade commercially available. Wistar albino rats obtained from the Industrial T o x i c o l o g y R e s e a r c h Centre, A n i m a l B r e e d i n g C o l o n y were maintained on a pellet diet (Hindustan Lever Laboratory Animal Feed, Bombay, India) and water ad llbltum. F e m a l e rats of p r o v e n f e r t i l i t y were mated (3:1) with adult males. On day one of pregnancy (confirmed by a p o s i t i v e v a g i n a l smear) the pregnant f e m a l e s were r a n d o m l y d i v i d e d into four groups and were housed in i n d i v i d u a l cages. On g e s t a t l o n a l day four Group I, II and III w e r e a d m i n i s t e r e d w i t h Fv, Cyp and Dt at a dose of I0, 15 and 7 mg/kg body weight r e s p e c t i v e l y in corn oll by oral g a v a g e from GD 5 to GD 21. G r o u p IV r e c e i v e d c o r n oll in an i d e n t i c a l m a n n e r . Progeny were e v a l u a t e d for behavioral teratogeneclty using the p r o c e d u r e d e s c r i b e d by V o r h e e s et al. (1979), d e s i g n e d to a s s e s s p h y s i c a l m i l e s t o n e s i.e. preweanlng and p o s t w e a n l n g tests. T h e s e i n c l u d e d pinna d e t a c h m e n t , incisor erruptlon, s u r f a c e righting, air righting, grip strength, auditory startle, ear opening, eye o p e n i n g and growth. M o t o r a c t i v i t y was tested in both the p r e w e a n e d and p o s t w e a n e d pups at day 21 and day 42 in a p h o t o a c t o m e t e r (Techno, India). R a t s were placed individually in the test a p p a r a t u s and their activity scores were recorded for a period of i0 minutes a c c o r d i n g to the m e t h o d of K u h n and V a n M a a n e r (1961). 403
At postnatal day t w e n t y two, five pups from each treatment group were decapitated by cervical dislocation. Brains were rapidly removed and brain regions i.e. pons m e d u l l a , h y p o t h a l a m u s , hippocampus, cerebellum, frontal c o r t e x and c o r p u s strlatum were dissected over dry ice a c c o r d i n g to the method of Glowlnskl and I v e r s o n (1966). Each brain part was homogenized (20% w/v) in cold 0.2 M perchloric acid using a Potter-Elvehjem homogenizer. The homogenates were k e p t at 5 ~ for 2 h and c e n t r i f u g e d at 6000 rpm for I0 minutes in a refrigerated centrifuge. The supernatant was c o l l e c t e d for polyamine estimation. P u t r e s c l n e , s p e r m l d l n e and s p e r m i n e w e r e s e p a r a t e d from the supernatant by thin layer chromatography and quantitatively estimated by the spectrofluorometrlc method of Seller and Lamberty (1975) with some modifications. Data w e r e s t a t i s t i c a l l y a n a l y s e d by s t u d e n t ' s as described by F i s c h e r (1950) and P < c o n s i d e r e d to be s i g n i f i c a n t . RESULTS
AND
't' 0.05
test was
DISCUSSION
No gross a b n o r m a l i t y was n o t e d in any of the treatment groups, however at h i g h e r doses i.e. 20, 30 and 15 mg/kg of Fv, Cyp and Dt r e s p e c t i v e l y , c a n n i b a l i s m was observed in a few dams in each treatment group accompanied w i t h f o e t a l r e s o r p t l o n s , loss in maternal and pup body weight. Dt e x p o s e d dams also showed vaginal b l e e d i n g , f o e t a l and m a t e r n a l d e a t h five days post treatment. S i m i l a r o b s e r v a t i o n s were made by Gupta (1988) w i t h Cyp (30 m g / k g ) . In a b s e n c e of any o t h e r t e r a t o g e n i c l t y or d e v e l o p m e n t a l t o x i c i t y data, we selected dose levels w h i c h did not cause any maternal toxicity. Body weights of the progeny showed no s i g n i f i c a n t change, h o w e v e r , i n d i v i d u a l w e i g h t s of some brain regions w e r e r e d u c e d by Dt, i n c r e a s e d by Cyp, while Fv had no effect (data not shown). Fv or Cyp treatment did not adversely affect the length of gestation and maternal body weight. However, Dt exposure r e s u l t e d in f o e t a l r e s o r p t l o n s and death of neonates and m e a n n u m b e r of pups in each litter was small in c o m p a r i s o n to v e h i c l e c o n t r o l s . Table 1 & 2 shows the effect of p r e n a t a l exposure to Fv, Cyp or Dt on the v a r i o u s d e v e l o p m e n t a l events in rat pups. All the three pyrethrolds significantly delayed the onset of fur d e v e l o p m e n t , incisor erruptlon, eye and ear o p e n i n g in c o m p a r i s o n to respective controls. Also the d e v e l o p m e n t of surface righting, r e f l e x and g r i p s t r e n g t h w e r e s i g n i f i c a n t l y r e d u c e d in the Fv and Dt e x p o s e d p r o g e n y , w h i l e only the surface righting reflex was altered by c y p e r m e thrln. 404
Table I. Effect of p r e n a t a l f e n v a l e r a t e ~ and d e l t a m e t h r i ~ e x p o s u r e on m o r p h o l o g i c a l events of rat pups Day Events
Occurrence
monitored Corn
P r i m a r y coat downy fur Incisor
of
cypermethrin b developmental
of
5
erruption
Development Ear
opening
Eye
opening
of
oll
fur
7-8
Fv
Cyp
5-6
7*
9-10"
Dt
6-7*
9-10"
i0"
9
9-10
i0-II*
i0-Ii*
Ii
12
12-14"
12-13"
15-17"
14-16*
15-16"
13-14
a , b , c (10,15,7 m g / k g body w e i g h t ) from GD-5 to GD-21, PO V a l u e s are m e a n of ten pups r a n d o m l y s e l e c t e d from ten litter in e a c h group. *p < 0.05 differs s i g n i f i c a n t l y f r o m c o n t r o l s ( S t u d e n t ' s 't' test) a
T a b l e 2. E f f e c t of p r e n a t a l and deltamethri~ exposure d e v e l o p m e n t of rat pups
fenvalerate, on locomotor
Day Events
of O c c u r r e n c e
monitored Corn
Forward
locomotion
Surface
righting
Grip
cyp erme thrin b and reflex
strength
Spontaneous locomotor activity ( c o u n t s per
: day 21 : : day 42 minute)
oil
I0
Fv
10-12
Cyp
10-12 10-13"
Dt
12"
9-10
9-10
12-13"
14
15-17"
14"
37.7+2.0
34+1.8
34+1.6
25.5+1.5"
40.0+2.2
39+2.0
42+1.4
22.0+1.2"
17"
a,b,c (10,15,7 m g / k g b o d y w e l g h t ~ from GD-5 to GD-21, PO V a l u e s are m e a n of ten pups r a n d o m l y s e l e c t e d from ten litter in e a c h group. *p < 0.05 differs s i g n i f i c a n t l y from c o n t r o l s ( S t u d e n t ' s 't' test) 405
Spontaneous motor activity monitored preweaning and postweaning, did not show any e f f e c t of Cyp or Fv but Dt s i g n i f i c a n t l y r e d u c e d the m o t o r a c t i v i t y at day 21 and 42 in c o m p a r i s o n to c o n t r o l s . Figure 1 shows the c o n t e n t of p o l y a m i n e s in different brain r e g i o n s in Fv e x p o s e d rat. Spermidlne level in corpus s t r i a t u m and c e r e b e l l u m i n c r e a s e d by 331% and 889% r e s p e c t i v e l y w h i l e p u t r e s c i n e l e v e l s d e c r e a s e d in all other b r a i n r e g i o n s e x c e p t the h y p o t h a l a m u s where it increased over c o n t r o l v a l u e s by 202%. Spermlne levels i n c r e a s e d in h y p o t h a l a m u s by 118%, hippocampus by 113% and pons m e d u l l a by 58% and d e c r e a s e d in c o r p u s s t r i a t u m by 66% w h i l e no s i g n i f i c a n t c h a n g e in frontal cortex and c e r e b e l l u m was e v i d e n t . The c h a n g e in the concentcation of p o l y a m i n e in d i f f e r e n t b r a i n regions in Cyp exposed rats is shown in Figure 2. Cyp significantly elevated the levels of all the three amines in all brain regions with the exception of putrescine levels in pons m e d u l l a w i t h a decrease of 74% over c o n t r o l s . The p e r c e n t i n c r e a s e in s p e r m i d i n e level over c o n t r o l , was 265% in pons m e d u l l a , 244% in corpus s t r i a t u m , 198% in h y p o t h a l a m u s , 116% in cerebellum, 91% in f r o n t a l c o r t e x and 76% in hippocampus. The s p e r m l n e l e v i e s a l s o s h o w e d s i g n i f i c a n t i n c r e a s e in its c o n c e n t r a t i o n in d i f f e r e n t b r a i n r e g i o n s viz. 362% in pons m e d u l l a , 292% in c o r p u s s t r l a t u m and 199% in hypothalamus. An o v e r a l l d e c r e a s e in the levels of all three polyamlnes was o b s e r v e d in most of the brain regions f o l l o w i n g Dt e x p o s u r e ( F i g u r e 3). The levels of all the t h r e e p o l y a m i n e s s i g n i f i c a n t l y d e c r e a s e d in hypothalamus and pons medulla while putresclne concentration s h o w e d an i n c r e a s e in f r o n t a l c o r t e x by 42%, h i p p o c a m p u s by 58% and c e r e b e l l u m by 24% over the controls. The s p e r m l d l n e and s p e r m i n e l e v e l s d e c r e a s e d in all b r a i n r e g i o n s e x c e p t in c o r p u s s t r l a t u m showing 38% and 19% i n c r e a s e r e s p e c t i v e l y over the control value 9 In the c e n t r a l n e r v o u s s y s t e m p o l y a m i n e s are p r e s e n t in high concentrations with considerable regional variability at c r i t i c a l s t a g e s of d e v e l o p m e n t (Shaw and Pateman 1973). The s p e c i f i c role of polyamlnes in m o d u l a t i o n of c e n t r a l s y n a p t i c t r a n s m i s s i o n , regeneration of the damaged neuronal circuitary following ischemic and other kinds of CNS injuries is well d o c u m e n t e d ( F e r c h m i n and E t e r o v l e 1987; P a s c h e n et al. 1987). Our data d e m o n s t r a t e s that e x p o s u r e to the three cyanopyrethroids viz. Fv, Cyp and Dt l e a d s to marked region s p e c i f i c a l t e r a t i o n s in p o l y a m i n e levels. The most a f f e c t e d areas w e r e h y p o t h a l a m u s and hippocampus. However, these a l t e r a t i o n s w e r e not u n i f o r m and the diversity could be explained on the basis of differential chemobiodynamlc p o t e n t i a l of the three 406
500-
30C
2
w el
w r
100
p::e o o 300
~ w
500
1000'
i
[ ] SPM
[ ] SPD
BII PUT
PUT
[]
SPM
[ ] sPo
N
IO0-
[]
[] SPM
SPD
PUT-putrescine, SPD-spermidine, SPM-spermine, CON-control, FC-frontal cortex, CS-corpus striatum, HPhippocampus, PM-pons medulla, HY-hypothalamus, CB-cerebellum
Figure 1 , 2 , 3 . Effect of f e n v a l e r a t e ( 1 ) , cypermethrin (2) and deltamethrin (3) on regional distribution of p o l y amines in brain.
3
uJ o.
u
~ z
o I.)
t~
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200-
IBI PUT
pyrethrolds in d i s c r e t e b r a i n r e g i o n s due to differences in t h e i r d e t o x i c a t l o n pathway. It is reported that although p r e t r e a t m e n t w i t h Cyp and Fv does not i n c r e a s e the a c t i v i t y of h e p a t i c m i x e d f u n c t i o n o x l d a s e system, since d e t o x l f l c a t i o n is mainly by carboxyl e s t e r a s e s , on the c o n t r a r y , Dt i n h i b i t s the drug metab o l i z i n g e n z y m e s ( A n a d o n et al. 1991). H e n c e the mode of n e u r o t o x l c a c t i o n of Fv and Cyp may d i f f e r f r o m Dt due to d i s t i n c t i o n in their p h a r m a c o k i n e t l c s as e v i d e n t from d i f f e r e n t i a l e f f e c t s on r e g i o n a l b r a i n polyamlne levels in g r o w i n g rats. The d e p l e t i o n of polyamlne levels in locallsed areas of the brain by Dt or increase by Fv and Cyp as o b s e r v e d in the present investigation, suggests a derangement in the early stages of the cell cycle when cell division and p r o l i f e r a t i o n are taking place. The o b s e r v e d d e l a y in morphological development, reflex ontogeny and motor function which are more pronounced in Dt exposed p r o g e n y , i m p l i e s d e l a y e d m a t u r a t i o n of c e r e b r a l cortex of these offsprings, since successful righting is dependent on the d e v e l o p m e n t of f u n c t i o n a l motor and sensory systems. P o l y a m l n e s also m e d i a t e c a l c i u m h o m e o s t a s i s , essential for the f u n c t i o n a l i n t e g r i t y of the cell ( K o e n l g et al. 1988). A d e p r e s s i o n or i n c r e a s e in p o l y a m l n e levels may p e r t u r b m e m b r a n e d e p o l a r i z a t i o n , f i r i n g rate of n e u r o n s and t r a n s d u c t l o n of i m p u l s e s a c r o s s the s y n a p s e l e a d i n g to d i s t u r b e d p a t h o p h y s l o l o g y of the n e u r o n . F r o m the f o r e g o i n g d i s c u s s i o n we can c o n c l u d e that Fv, Cyp and Dt i n d u c e d d e v i a t i o n in s o m a t o - s e n s o r y indices and o p t i m a l c o n c e n t r a t i o n of p o l y a m l n e s in localized brain areas, m a y p a r t l y a c c o u n t for the perturbations in the n o r m a l o n t o g e n y of neurotransmltter system(s) and behavioural deficits, causing a further delay in c e r e b r a l m a t u r a t i o n and f u n c t i o n s .
Acknowledgment.
The a u t h o r s thank Prof. P.K. Ray, Director, ITRC, L u c k n o w for his a c t i v e s u p p o r t during the course of this study. Financial assistance of Indian C o u n c i l of M e d i c a l R e s e a r c h , N e w Delhi, India for s u p p o r t i n g this s t u d y is t h a n k f u l l y a c k n o w l e d g e d .
REFERENCES Aldrldge WN (1990) An a s s e s s m e n t of the toxicological properties of p y r e t h r o l d s and their neurotoxiclty. CRC C r i t i c a l R e v i e w of T o x i c o l o g y 21: 8 9 - 1 0 4 Anadon A, M a r t l n e z - L a r r a n g a MR, Diaz MJ, Bringas P, Fernandez MC (1991) Effect of deltamethrin on antlpyrlne p h a r m a c o k i n e t i c s and m e t a b o l i s m in rat. A r c h T o x l c o l 65: 1 5 6 - 1 5 9 408
Ferchmin PA, E t e r o v i e VA ( 1 9 8 7 ) R o l e o f p o l y a m i n e s in experlence-dependent brain plasticity. Pharmacol B i o c h e m Behav 2 6 : 3 4 1 - 3 4 9 Fischer RA (1950) S t a t i s t i c a l methods for research w o r k e r s , llth ed. O l i v e r and Boyd, London Glowinski J, Iverson (1966) R e g i o n a l studies of the catecholamlnes in rat brain. J N e u r o c h e m 13: 655669 Gupta PK (1988) C y p e r m e t h r i n - A pyrethrold insecticide in Gupta PK, P r a k a s h R (ed) A d v a n c e s in Toxicology and E n v i r o n m e n t a l Health, J a g m a n d e r Book Agency, New Delhi, pp 81 Husaln R, Gupta A, K h a n n a VK, Seth PK (1991) N e u r o t o x i cological effects of a pyrethroid formulation, fenvalerate in rats. Res Commun Chem Pathol P h a r m a c o l 73: 111-114 K o e n i g H, G o l d s t o n e A, Lu Ch Y, lqbal Z, Fau CC, Trout JJ (1988) Polyamines, hormone receptors and calcium f l u x e s : i n B a c h a r a c h U, B e i m e r YM (ed) The P h y s i o l o g y of Polyamines, CRC Press, Boco Raton, Florida Kuhn WL, Van Maaner (1961) Central nervous system effects of phthalidamide. J P h a r m a c o l Exp Ther 134: 60-68 Paschen W, S e h m i d t - K a s t u e r R, DJuricic B, Meese C, Linn F, H o s s m a n KA (1987) P o l y a m l n e changes in r e v e r s i b l e cerebral ischemia. J N e u r o c h e m 49: 35-37 Russel DH, Durie BG (1978) Polyamlnes as biochemical markers of normal and m a l i g n a n t growth, Progress in Cancer R e s e a r c h and Therapy, vol 8, Raven Press~ New York Seiler N, L a m b e r t y V (1975) I n t e r a c t i o n between polyamine and nucleic acids, changes of polyamlnes and n u c l e i c acids in developing rats brain. J Neurochem 24 : I-I 6 Shaw GG, P a t e m a n AJ (1973) The regional d i s t r i b u t i o n of polyamlnes spermldine and spermlne in brain. J N e u r o c h e m 20: 1 2 2 5 - 1 2 3 0 Tabor H, Tabor CW (1983) P o l y a m l n e s in: Methods in enzymology vol 94, A c a d e m i c Press, New York, pp 497 Verschoyle RD, A l d r i d g e WN (1980) Structure-actlvity relationships of some pyrethrolds in rats. Arch Toxicol 45: 325-329 Vijverberg HPM, Bercken J (1990) Neurotoxlcologlcal effects and the mode of action of pyrethrold insecticides. CRC Critical R e v i e w of T o x i c o l o g y 21: 105-126 Vorhees CV, B u t c h e r RE, Brunner RL, Sobotka TJ (1979) A developmental test battery for neurobehavloral t o x i c i t y in rats: A p r e l i m i n a r y analysis using monosodium glutamate, c a l c i u m c a r r a g e e n a n and hydroxyurea. T o x i c o l Appl P h a r m a c o l 5 0 : 2 6 7 - 2 8 2 ~ceivedNovemberll, l~l;accepted M~chll, l~2.
409
i
Snvironmental Contamination and Toxicology
Differential Responses of Regional Brain Polyamines Following In Utero Exposure to Synthetic Pyrethroid Insecticides: A Preliminary Report Raghib Husain, Madhu Malaviya, Prahlad K. Seth, and Raushan Husain Industrial Toxicology Research Centre, Post Box 80, Mahatma Gandhi Marg, Lucknow-226 001, India Synthetic p y r e t h r o i d s are p o t e n t i n s e c t i c i d e s of high bloefficacy. T h e y are now b e i n g e x t e n s i v e l y u s e d for pest management in a g r i c u l t u r a l p r a c t i c e s in India. M a m m a l i a n c e n t r a l n e r v o u s s y s t e m is the p r i m a r y target for the toxic a c t i o n of the synthetic pyrethroids, which are c l a s s i f i e d into two c a t e g o r i e s a c c o r d i n g to the s i g n s and s y m p t o m s of t o x i c i t y e x h i b i t e d i.e. TypeI (T-Syndrome) and Type-ll (CS-Syndrome) (Aldridge 1990; V i j v e r b e r g and B e r c k e n 1990). The T-syndrome is characterlsed by hyperexcltation, tremor and c o n v u l s i o n s w h i l e C S - s y n d r o m e by an i n i t i a l h y p e r a c t i v e behavlour, facial tremor, profuse salivation and spontaneous choreathetosis. It is now w e l l e s t a b l i s h e d that pyrethroids exert their neurotoxic effects primarily by m o d i f y i n g s o d i u m g a t i n g k i n e t i c s and the neurotoxic p o t e n t i a l of T y p e - l l is c h i e f l y due to the o~-cyano-3-phenoxybenzyl alcohol moiety (Verschoyle and Aldridge 1980). Our e a r l i e r s t u d i e s w i t h fenvalerate g i v e n o r a l l y to adult rats s h o w e d m a r k e d i n h i b i t i o n in regional brain levels of catecholamines and their m e t a b o l i t e s ( H u s a i n et al. 1991). The e x t e n s i v e and i n j u d i c i o u s use of i n s e c t i c i d e s poses d i r e c t or i n d i r e c t h e a l t h h a z a r d , to man and live s t o c k as well as leads to environmental contamination. E x p o s u r e of p r e g n a n t w o m e n at the m a n u f a c t u r i n g sites, in the f i e l d s or t h r o u g h environmental contamination poses h e a l t h risk to d e v e l o p i n g h u m a n fetus. Insplte of numerous s t u d i e s c o n f i r m i n g that pyrethroids are n e u r o t o x i c to the adult n e r v o u s s y s t e m , l i t t l e is k n o w n about their perinatal effects. The fetal brain is e s p e c i a l l y v u l n e r a b l e to c h e m i c a l s w h i c h r e a d i l y cross the p l a c e n t a l b a r r i e r d u r i n g b r a i n s p u r t p e r i o d . Since Send above
reprint address.
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during this time c e l l u l a r d e v e l o p m e n t and ontogenesis of various neurotransmitter system(s) progresses r a p i d l y and o f t e n the e f f e c t s i n d u c e d by the chemicals or drugs lead to f u n c t i o n a l and behavioural deficits w h i c h a p p e a r m u c h later in c h i l d h o o d . The p r e s e n t s t u d y was t h e r e f o r e a i m e d to investigate the in utero e f f e c t s of three t y p e - l l p y r e t h r o l d s vlz. Fenvalerate (Fv), C y p e r m e t h r l n (Cyp) and Deltamethrln (Dt) on the early brain development. Levels of polyamines reported to be closely associated with g r o w t h and d i f f e r e n t i a t i o n (Tabor and Tabor 1983) and the r e g u l a t i o n of DNA, RNA and p r o t e i n synthesls in many g r o w i n g systems ( B a c h r a c h 1973), w e r e s t u d i e d in s p e c l f [ c r e g i o n s of rat brain. MATERIALS
AND
METHODS
Putresclne dihydrochloride, spermidine trihydrochloride, spermlne tetrahydrochlorlde and d a n s y l chloride were obtained from Sigma C h e m i c a l s Co., St. Louis, USA. E m u l s i f i a b l e c o n c e n t r a t e s of f e n v a l e r a t e , deltamethrin and c y p e r m e t h r l n in 2 0 % , 2 . 8 % and 25% EC formulations, w/w r e s p e c t i v e l y were o b t a i n e d from Shroff Industrial Chemicals, Gujarat, India. All other reagents and solvents used w e r e of the highest analytical grade commercially available. Wistar albino rats obtained from the Industrial T o x i c o l o g y R e s e a r c h Centre, A n i m a l B r e e d i n g C o l o n y were maintained on a pellet diet (Hindustan Lever Laboratory Animal Feed, Bombay, India) and water ad llbltum. F e m a l e rats of p r o v e n f e r t i l i t y were mated (3:1) with adult males. On day one of pregnancy (confirmed by a p o s i t i v e v a g i n a l smear) the pregnant f e m a l e s were r a n d o m l y d i v i d e d into four groups and were housed in i n d i v i d u a l cages. On g e s t a t l o n a l day four Group I, II and III w e r e a d m i n i s t e r e d w i t h Fv, Cyp and Dt at a dose of I0, 15 and 7 mg/kg body weight r e s p e c t i v e l y in corn oll by oral g a v a g e from GD 5 to GD 21. G r o u p IV r e c e i v e d c o r n oll in an i d e n t i c a l m a n n e r . Progeny were e v a l u a t e d for behavioral teratogeneclty using the p r o c e d u r e d e s c r i b e d by V o r h e e s et al. (1979), d e s i g n e d to a s s e s s p h y s i c a l m i l e s t o n e s i.e. preweanlng and p o s t w e a n l n g tests. T h e s e i n c l u d e d pinna d e t a c h m e n t , incisor erruptlon, s u r f a c e righting, air righting, grip strength, auditory startle, ear opening, eye o p e n i n g and growth. M o t o r a c t i v i t y was tested in both the p r e w e a n e d and p o s t w e a n e d pups at day 21 and day 42 in a p h o t o a c t o m e t e r (Techno, India). R a t s were placed individually in the test a p p a r a t u s and their activity scores were recorded for a period of i0 minutes a c c o r d i n g to the m e t h o d of K u h n and V a n M a a n e r (1961). 403
At postnatal day t w e n t y two, five pups from each treatment group were decapitated by cervical dislocation. Brains were rapidly removed and brain regions i.e. pons m e d u l l a , h y p o t h a l a m u s , hippocampus, cerebellum, frontal c o r t e x and c o r p u s strlatum were dissected over dry ice a c c o r d i n g to the method of Glowlnskl and I v e r s o n (1966). Each brain part was homogenized (20% w/v) in cold 0.2 M perchloric acid using a Potter-Elvehjem homogenizer. The homogenates were k e p t at 5 ~ for 2 h and c e n t r i f u g e d at 6000 rpm for I0 minutes in a refrigerated centrifuge. The supernatant was c o l l e c t e d for polyamine estimation. P u t r e s c l n e , s p e r m l d l n e and s p e r m i n e w e r e s e p a r a t e d from the supernatant by thin layer chromatography and quantitatively estimated by the spectrofluorometrlc method of Seller and Lamberty (1975) with some modifications. Data w e r e s t a t i s t i c a l l y a n a l y s e d by s t u d e n t ' s as described by F i s c h e r (1950) and P < c o n s i d e r e d to be s i g n i f i c a n t . RESULTS
AND
't' 0.05
test was
DISCUSSION
No gross a b n o r m a l i t y was n o t e d in any of the treatment groups, however at h i g h e r doses i.e. 20, 30 and 15 mg/kg of Fv, Cyp and Dt r e s p e c t i v e l y , c a n n i b a l i s m was observed in a few dams in each treatment group accompanied w i t h f o e t a l r e s o r p t l o n s , loss in maternal and pup body weight. Dt e x p o s e d dams also showed vaginal b l e e d i n g , f o e t a l and m a t e r n a l d e a t h five days post treatment. S i m i l a r o b s e r v a t i o n s were made by Gupta (1988) w i t h Cyp (30 m g / k g ) . In a b s e n c e of any o t h e r t e r a t o g e n i c l t y or d e v e l o p m e n t a l t o x i c i t y data, we selected dose levels w h i c h did not cause any maternal toxicity. Body weights of the progeny showed no s i g n i f i c a n t change, h o w e v e r , i n d i v i d u a l w e i g h t s of some brain regions w e r e r e d u c e d by Dt, i n c r e a s e d by Cyp, while Fv had no effect (data not shown). Fv or Cyp treatment did not adversely affect the length of gestation and maternal body weight. However, Dt exposure r e s u l t e d in f o e t a l r e s o r p t l o n s and death of neonates and m e a n n u m b e r of pups in each litter was small in c o m p a r i s o n to v e h i c l e c o n t r o l s . Table 1 & 2 shows the effect of p r e n a t a l exposure to Fv, Cyp or Dt on the v a r i o u s d e v e l o p m e n t a l events in rat pups. All the three pyrethrolds significantly delayed the onset of fur d e v e l o p m e n t , incisor erruptlon, eye and ear o p e n i n g in c o m p a r i s o n to respective controls. Also the d e v e l o p m e n t of surface righting, r e f l e x and g r i p s t r e n g t h w e r e s i g n i f i c a n t l y r e d u c e d in the Fv and Dt e x p o s e d p r o g e n y , w h i l e only the surface righting reflex was altered by c y p e r m e thrln. 404
Table I. Effect of p r e n a t a l f e n v a l e r a t e ~ and d e l t a m e t h r i ~ e x p o s u r e on m o r p h o l o g i c a l events of rat pups Day Events
Occurrence
monitored Corn
P r i m a r y coat downy fur Incisor
of
cypermethrin b developmental
of
5
erruption
Development Ear
opening
Eye
opening
of
oll
fur
7-8
Fv
Cyp
5-6
7*
9-10"
Dt
6-7*
9-10"
i0"
9
9-10
i0-II*
i0-Ii*
Ii
12
12-14"
12-13"
15-17"
14-16*
15-16"
13-14
a , b , c (10,15,7 m g / k g body w e i g h t ) from GD-5 to GD-21, PO V a l u e s are m e a n of ten pups r a n d o m l y s e l e c t e d from ten litter in e a c h group. *p < 0.05 differs s i g n i f i c a n t l y f r o m c o n t r o l s ( S t u d e n t ' s 't' test) a
T a b l e 2. E f f e c t of p r e n a t a l and deltamethri~ exposure d e v e l o p m e n t of rat pups
fenvalerate, on locomotor
Day Events
of O c c u r r e n c e
monitored Corn
Forward
locomotion
Surface
righting
Grip
cyp erme thrin b and reflex
strength
Spontaneous locomotor activity ( c o u n t s per
: day 21 : : day 42 minute)
oil
I0
Fv
10-12
Cyp
10-12 10-13"
Dt
12"
9-10
9-10
12-13"
14
15-17"
14"
37.7+2.0
34+1.8
34+1.6
25.5+1.5"
40.0+2.2
39+2.0
42+1.4
22.0+1.2"
17"
a,b,c (10,15,7 m g / k g b o d y w e l g h t ~ from GD-5 to GD-21, PO V a l u e s are m e a n of ten pups r a n d o m l y s e l e c t e d from ten litter in e a c h group. *p < 0.05 differs s i g n i f i c a n t l y from c o n t r o l s ( S t u d e n t ' s 't' test) 405
Spontaneous motor activity monitored preweaning and postweaning, did not show any e f f e c t of Cyp or Fv but Dt s i g n i f i c a n t l y r e d u c e d the m o t o r a c t i v i t y at day 21 and 42 in c o m p a r i s o n to c o n t r o l s . Figure 1 shows the c o n t e n t of p o l y a m i n e s in different brain r e g i o n s in Fv e x p o s e d rat. Spermidlne level in corpus s t r i a t u m and c e r e b e l l u m i n c r e a s e d by 331% and 889% r e s p e c t i v e l y w h i l e p u t r e s c i n e l e v e l s d e c r e a s e d in all other b r a i n r e g i o n s e x c e p t the h y p o t h a l a m u s where it increased over c o n t r o l v a l u e s by 202%. Spermlne levels i n c r e a s e d in h y p o t h a l a m u s by 118%, hippocampus by 113% and pons m e d u l l a by 58% and d e c r e a s e d in c o r p u s s t r i a t u m by 66% w h i l e no s i g n i f i c a n t c h a n g e in frontal cortex and c e r e b e l l u m was e v i d e n t . The c h a n g e in the concentcation of p o l y a m i n e in d i f f e r e n t b r a i n regions in Cyp exposed rats is shown in Figure 2. Cyp significantly elevated the levels of all the three amines in all brain regions with the exception of putrescine levels in pons m e d u l l a w i t h a decrease of 74% over c o n t r o l s . The p e r c e n t i n c r e a s e in s p e r m i d i n e level over c o n t r o l , was 265% in pons m e d u l l a , 244% in corpus s t r i a t u m , 198% in h y p o t h a l a m u s , 116% in cerebellum, 91% in f r o n t a l c o r t e x and 76% in hippocampus. The s p e r m l n e l e v i e s a l s o s h o w e d s i g n i f i c a n t i n c r e a s e in its c o n c e n t r a t i o n in d i f f e r e n t b r a i n r e g i o n s viz. 362% in pons m e d u l l a , 292% in c o r p u s s t r l a t u m and 199% in hypothalamus. An o v e r a l l d e c r e a s e in the levels of all three polyamlnes was o b s e r v e d in most of the brain regions f o l l o w i n g Dt e x p o s u r e ( F i g u r e 3). The levels of all the t h r e e p o l y a m i n e s s i g n i f i c a n t l y d e c r e a s e d in hypothalamus and pons medulla while putresclne concentration s h o w e d an i n c r e a s e in f r o n t a l c o r t e x by 42%, h i p p o c a m p u s by 58% and c e r e b e l l u m by 24% over the controls. The s p e r m l d l n e and s p e r m i n e l e v e l s d e c r e a s e d in all b r a i n r e g i o n s e x c e p t in c o r p u s s t r l a t u m showing 38% and 19% i n c r e a s e r e s p e c t i v e l y over the control value 9 In the c e n t r a l n e r v o u s s y s t e m p o l y a m i n e s are p r e s e n t in high concentrations with considerable regional variability at c r i t i c a l s t a g e s of d e v e l o p m e n t (Shaw and Pateman 1973). The s p e c i f i c role of polyamlnes in m o d u l a t i o n of c e n t r a l s y n a p t i c t r a n s m i s s i o n , regeneration of the damaged neuronal circuitary following ischemic and other kinds of CNS injuries is well d o c u m e n t e d ( F e r c h m i n and E t e r o v l e 1987; P a s c h e n et al. 1987). Our data d e m o n s t r a t e s that e x p o s u r e to the three cyanopyrethroids viz. Fv, Cyp and Dt l e a d s to marked region s p e c i f i c a l t e r a t i o n s in p o l y a m i n e levels. The most a f f e c t e d areas w e r e h y p o t h a l a m u s and hippocampus. However, these a l t e r a t i o n s w e r e not u n i f o r m and the diversity could be explained on the basis of differential chemobiodynamlc p o t e n t i a l of the three 406
500-
30C
2
w el
w r
100
p::e o o 300
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500
1000'
i
[ ] SPM
[ ] SPD
BII PUT
PUT
[]
SPM
[ ] sPo
N
IO0-
[]
[] SPM
SPD
PUT-putrescine, SPD-spermidine, SPM-spermine, CON-control, FC-frontal cortex, CS-corpus striatum, HPhippocampus, PM-pons medulla, HY-hypothalamus, CB-cerebellum
Figure 1 , 2 , 3 . Effect of f e n v a l e r a t e ( 1 ) , cypermethrin (2) and deltamethrin (3) on regional distribution of p o l y amines in brain.
3
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o I.)
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IBI PUT
pyrethrolds in d i s c r e t e b r a i n r e g i o n s due to differences in t h e i r d e t o x i c a t l o n pathway. It is reported that although p r e t r e a t m e n t w i t h Cyp and Fv does not i n c r e a s e the a c t i v i t y of h e p a t i c m i x e d f u n c t i o n o x l d a s e system, since d e t o x l f l c a t i o n is mainly by carboxyl e s t e r a s e s , on the c o n t r a r y , Dt i n h i b i t s the drug metab o l i z i n g e n z y m e s ( A n a d o n et al. 1991). H e n c e the mode of n e u r o t o x l c a c t i o n of Fv and Cyp may d i f f e r f r o m Dt due to d i s t i n c t i o n in their p h a r m a c o k i n e t l c s as e v i d e n t from d i f f e r e n t i a l e f f e c t s on r e g i o n a l b r a i n polyamlne levels in g r o w i n g rats. The d e p l e t i o n of polyamlne levels in locallsed areas of the brain by Dt or increase by Fv and Cyp as o b s e r v e d in the present investigation, suggests a derangement in the early stages of the cell cycle when cell division and p r o l i f e r a t i o n are taking place. The o b s e r v e d d e l a y in morphological development, reflex ontogeny and motor function which are more pronounced in Dt exposed p r o g e n y , i m p l i e s d e l a y e d m a t u r a t i o n of c e r e b r a l cortex of these offsprings, since successful righting is dependent on the d e v e l o p m e n t of f u n c t i o n a l motor and sensory systems. P o l y a m l n e s also m e d i a t e c a l c i u m h o m e o s t a s i s , essential for the f u n c t i o n a l i n t e g r i t y of the cell ( K o e n l g et al. 1988). A d e p r e s s i o n or i n c r e a s e in p o l y a m l n e levels may p e r t u r b m e m b r a n e d e p o l a r i z a t i o n , f i r i n g rate of n e u r o n s and t r a n s d u c t l o n of i m p u l s e s a c r o s s the s y n a p s e l e a d i n g to d i s t u r b e d p a t h o p h y s l o l o g y of the n e u r o n . F r o m the f o r e g o i n g d i s c u s s i o n we can c o n c l u d e that Fv, Cyp and Dt i n d u c e d d e v i a t i o n in s o m a t o - s e n s o r y indices and o p t i m a l c o n c e n t r a t i o n of p o l y a m l n e s in localized brain areas, m a y p a r t l y a c c o u n t for the perturbations in the n o r m a l o n t o g e n y of neurotransmltter system(s) and behavioural deficits, causing a further delay in c e r e b r a l m a t u r a t i o n and f u n c t i o n s .
Acknowledgment.
The a u t h o r s thank Prof. P.K. Ray, Director, ITRC, L u c k n o w for his a c t i v e s u p p o r t during the course of this study. Financial assistance of Indian C o u n c i l of M e d i c a l R e s e a r c h , N e w Delhi, India for s u p p o r t i n g this s t u d y is t h a n k f u l l y a c k n o w l e d g e d .
REFERENCES Aldrldge WN (1990) An a s s e s s m e n t of the toxicological properties of p y r e t h r o l d s and their neurotoxiclty. CRC C r i t i c a l R e v i e w of T o x i c o l o g y 21: 8 9 - 1 0 4 Anadon A, M a r t l n e z - L a r r a n g a MR, Diaz MJ, Bringas P, Fernandez MC (1991) Effect of deltamethrin on antlpyrlne p h a r m a c o k i n e t i c s and m e t a b o l i s m in rat. A r c h T o x l c o l 65: 1 5 6 - 1 5 9 408
Ferchmin PA, E t e r o v i e VA ( 1 9 8 7 ) R o l e o f p o l y a m i n e s in experlence-dependent brain plasticity. Pharmacol B i o c h e m Behav 2 6 : 3 4 1 - 3 4 9 Fischer RA (1950) S t a t i s t i c a l methods for research w o r k e r s , llth ed. O l i v e r and Boyd, London Glowinski J, Iverson (1966) R e g i o n a l studies of the catecholamlnes in rat brain. J N e u r o c h e m 13: 655669 Gupta PK (1988) C y p e r m e t h r i n - A pyrethrold insecticide in Gupta PK, P r a k a s h R (ed) A d v a n c e s in Toxicology and E n v i r o n m e n t a l Health, J a g m a n d e r Book Agency, New Delhi, pp 81 Husaln R, Gupta A, K h a n n a VK, Seth PK (1991) N e u r o t o x i cological effects of a pyrethroid formulation, fenvalerate in rats. Res Commun Chem Pathol P h a r m a c o l 73: 111-114 K o e n i g H, G o l d s t o n e A, Lu Ch Y, lqbal Z, Fau CC, Trout JJ (1988) Polyamines, hormone receptors and calcium f l u x e s : i n B a c h a r a c h U, B e i m e r YM (ed) The P h y s i o l o g y of Polyamines, CRC Press, Boco Raton, Florida Kuhn WL, Van Maaner (1961) Central nervous system effects of phthalidamide. J P h a r m a c o l Exp Ther 134: 60-68 Paschen W, S e h m i d t - K a s t u e r R, DJuricic B, Meese C, Linn F, H o s s m a n KA (1987) P o l y a m l n e changes in r e v e r s i b l e cerebral ischemia. J N e u r o c h e m 49: 35-37 Russel DH, Durie BG (1978) Polyamlnes as biochemical markers of normal and m a l i g n a n t growth, Progress in Cancer R e s e a r c h and Therapy, vol 8, Raven Press~ New York Seiler N, L a m b e r t y V (1975) I n t e r a c t i o n between polyamine and nucleic acids, changes of polyamlnes and n u c l e i c acids in developing rats brain. J Neurochem 24 : I-I 6 Shaw GG, P a t e m a n AJ (1973) The regional d i s t r i b u t i o n of polyamlnes spermldine and spermlne in brain. J N e u r o c h e m 20: 1 2 2 5 - 1 2 3 0 Tabor H, Tabor CW (1983) P o l y a m l n e s in: Methods in enzymology vol 94, A c a d e m i c Press, New York, pp 497 Verschoyle RD, A l d r i d g e WN (1980) Structure-actlvity relationships of some pyrethrolds in rats. Arch Toxicol 45: 325-329 Vijverberg HPM, Bercken J (1990) Neurotoxlcologlcal effects and the mode of action of pyrethrold insecticides. CRC Critical R e v i e w of T o x i c o l o g y 21: 105-126 Vorhees CV, B u t c h e r RE, Brunner RL, Sobotka TJ (1979) A developmental test battery for neurobehavloral t o x i c i t y in rats: A p r e l i m i n a r y analysis using monosodium glutamate, c a l c i u m c a r r a g e e n a n and hydroxyurea. T o x i c o l Appl P h a r m a c o l 5 0 : 2 6 7 - 2 8 2 ~ceivedNovemberll, l~l;accepted M~chll, l~2.
409
