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Endosulfan induces small but significant changes in the levels of noradrenaline, dopamine and serotonin in the developing rat brain and deficits in the operant learning performance
IHICOIOGY ELSEVIER
Toxicology 91 (1994) 139-150
Endosulfan induces small but significant changes in the levels of noradrenaline, dopamine and serotonin in the developing rat brain and deficits in the operant learning performance Madepalli K. Lakshmana, Trichur R. Raju* Department of Neurophysiology, National Institute of Mental Health and Neuro Sciences, Bangalore-560 029, India
(Received 18 August 1993; accepted 21 November 1993)
Abstract
The organochlorine insecticide, endosulfan was administered (6 mg/kg body weight) to Wistar rat pups of both sexes by gastric intubation daily from post-natal days 2-25. Its effect on levels of noradrenaline (NA), dopamine (DA) and serotonin (5-HT) was assayed in olfactory bulb (OB), hippocampus (HI), visual cortex (VC), brainstem (BS) and cerebellum (CB) on days 10 and 25 using high-performance liquid chromatography (HPLC). The activity of acetylcholinesterase (ACHE) was also estimated in the same regions of the brain. Performance in operant conditioning for solid food reward was assessed in 25-day-old rats. NA levels were increased in OB (12%, P < 0.01) and BS (10%, P < 0.05) at 10 days of age and in HI (20%, P < 0.01) and CB (12%, P < 0.05) at 25 days of age. DA level was decreased in H1 at both 10 (42%, P < 0.001) and 25 (45%, P < 0.001) days. Serotonin levels were increased in OB (12%, P < 0.05), HI (41%, P < 0.001), VC (30%, P < 0.01) and BS (15%, P < 0.01) at 10 days of age but at 25 days, levels were decreased in BS (20%, P < 0.05) and CB (31%, P < 0.01). The activity of AChE was not different from the control groups in any of the regions studied. These data suggest that monoaminergic systems in the developing rat brain respond to endosulfan by undergoing something like a 'reorganization'. However, such changes do not ameliorate certain functional losses following the exposure to endosulfan as operant conditioning revealed deficits in acquisition as well as retention of memory. Abbreviations: ACHE, Aeetylcholine esterase; BS, Brainstem; CB, Cerebellum; DA, Dopamine; HI, Hippoeampus; 5-HT, 5-hydroxytryptamine; NA, Noradrenaline; OB, Olfactory bulb; VC, Visual cortex * Corresponding author.
0300-483X/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0300-483X(93)02772-Z
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Keywords:Chronic endosulfan exposure; Developing rat brain; Monoamines; ACHE; Operant conditioning; HPLC
1. Introduction
Endosulfan, an organochlorine insecticide of cyclodiene structure has substantially replaced the earlier persistent type of chlorinated compounds such as dichlorodiphenyltrichloro acetic acid (DDT) and benzene hexachloride (BHC) and is now much in use for agriculture and crop protection (WHO, 1984). Endosulfan has been detected in ambient air samples (Kutz et al., 1976), in precipitation in the Great Lakes (Strachan et al., 1980) and in cow's milk (Frank et al., 1979) though at lower concentrations. High levels of endosulfan residues have also been reported in tobacco smoke (Chopra et al., 1978; Cheng and Braun, 1977). Clinical signs of endosulfan toxicity in accidental poisoning (Terziev et al., 1974) and in workers employed in chemical factories (Tiberin et al., 1970), suggest the involvement of the central nervous system and include symptoms such as hyperactivity, irritability, disorientation, tremors and convulsions followed by death. Toxicity of endosulfan to experimental animals has been reported (Gupta and Gupta, 1979). Altered electrical activity of the brain has been found after endosulfan administration, in cats (Anand, 1980) and in rats (Anand et al., 1980). However, the mechanism by which endosulfan induces toxicity remains elusive. Zaidi et al. (1985) have implicated the serotonergic system in endosulfan neurotoxicity in neo-natal rats from binding studies using 5-hydroxytryptamine (5-HT) blocker, methysergide; this was correlated with behavioural changes. Chronic endosulfan exposure has been shown to inhibit 5-HT uptake (Anand et al., 1986a) which supports the above finding. On the other hand Anand et al. (1986b) have suggested the involvement of the cholinergic system in the mediation of endosulfan neurotoxicity. It is possible that endosulfan may mediate its neurotoxic effect by acting on different neurotransmitter systems influencing their synthesis, release, uptake and degradation. Ansari et al. (1987) studied the effect of acute endosulfan exposure on different neurotransmitters of brain after a single intraperitoneal (i.p.) dose in male rats. They showed alterations in the levels of noradrenaline (NA), dopamine (DA) and 5-HT but not acetylcholine (ACh). However, a very high dose, about half of the lethal dose, was used in this study. Since endosulfan exposure is mainly through food intake, at relatively lower concentrations, it is not known whether chronic consumption of endosulfan at lower doses for longer duration could have similar effects. The possible effect of endosulfan on the developing nervous system, which is more vulnerable to external insults than the adult brain is also not yet studied. Accordingly, the present study was designed to investigate the effect of endosulfan consumption in a lower dose during post-natal brain growth period on the levels of NA, DA and 5-HT and on the activity of acetylcholinesterase (ACHE). As the accumulation of endosulfan has been shown to be uneven in the central nervous system (Gupta, 1978) these parameters were estimated in various regions of developing rat brain. In addition, rats were also subjected to operant conditioning, a paradigm used for testing learning and memory.
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2. Materials and methods
2.1. Materials
Endosulfan (6,7,8,9,10,10-hexachloro- 1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3benzodioxa-thiepin-3-oxide) obtained from Excel Co., Bombay, India, was of technical grade (96%). Standards such as norepinephrine bitartrate salt, dopamine hydrochloride, 5-hydroxytryptamine hydrochloride, isoproterenol hydrochloride and heptane sulfonic acid for monoamine estimations and acetylthiocholine iodide, dithiobisnitrobenzoate (DTNB) for AChE assay were all obtained from Sigma Chemical Co., St. Louis, MO, USA. Methanol of HPLC grade was obtained from Spectrochemicals, Bombay, India. Ethylenediaminetetraacetic acid (EDTA), sodium acetate, sodium bicarbonate, orthophosphoric acid and perchloric acid (70%) of analytical grade were all purchased from a local supplier, BDH, Bombay, India. Doubledistilled water was used for preparing all solutions. 2.2. Animals
Female Wistar rats were caged individually after confirmation of conception. Litter size was maintained at eight pups per mother. Rat pups of both sexes were allowed to be with mother throughout the day during weaning and thereafter had ad libitum access to water and a semi-synthetic balanced diet (18% casein, 78% wheat) (Mascarenhas et al., 1984) which contains all vitamins and minerals for the normal growth of rats. The room temperature was 25-26°C and 12- h light and dark cycles were strictly enforced in a fully ventilated room. 2.3. Experimental procedure
As shown in Fig. 1, the preliminary experiments in our laboratory revealed 8% inhibition of Na +, K + ATPase activity in rat brain microsomes as determined by the method of Davis (1970) at 10-6 M endosulfan. The presently observed Na+,K + ATPase inhibition was small at 10-6M (equivalent to 4.1 mg/kg body weight). Also, in a previous study, since no toxic symptoms were observed in rats at <__5 mg/kg body weight of endosulfan by oral intubation (Garg et al., 1980), in the present study 1.5 times this dosage was used. A group of experimental rats, were fed endosulfan (6 mg/kg body weight) (dissolved in peanut oil) daily between 09.00-10.00 h. Control groups of rat pups received equal amounts of peanut oil alone by the same route. Rat pups from control and experimental groups were sacrificed by decapitation on day 10 (n = 6 in each group) and day 25 (n = 6 in each group). Brains were removed immediately and different brain regions were dissected on an ice-cold Petri dish as described by Lindgren et al. (1982). 2.4. Monoamine estimations
The HPLC system consisted of a delivery pump (model ERC-8710, Erma Optical Works, Tokyo, Japan), a reverse-phase analytical column, Ultracarb 3 /~m ODS,
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M.K. Lakshmana, T.R. Raju / Toxicology 91 (1994) 139-150 100 O_
~- 80 i
+x-" ÷4
z
60
C
0
~_- 40 e-
~ 20 n i
166 Endosulfan
i
10-5
i
10-4
Concentration ( M )
Fig. 1. Inhibition of Na+K+-ATPase by endosulfan in rat brain microsomes. The ATPase activity was assayed by the liberation of Pi from the substrate ATP in 15 min at pH 7.5 and 35°C. Inhibition is expressed relative to a solvent control. Each point represents the mean ( ± S.E.) of six independent experiments performed on different days with brain microsomes.
150 × 4.6 mm (Phenomenex, Torrance, CA, USA), a guard column, Ultracarb 3 t~m ODS, 30 x 4.6 mm (phenomenex, Torrance, CA, USA), a degasser (model ERC 3310, ERMA INC, Tokyo, Japan), a recorder (Sekonic SS 100F) and a fluorescence spectrophotometer (Hitachi, model 650-40, Japan) for detection. Samples were prepared and analysed by a slightly modified procedure of Murai et al. (1988). The modified mobile phase, pH 3.92, consisted of sodium acetate 0.02 M, methanol 16% (v/v), heptane sulfonic acid 0.1375% (w/v) and EDTA 0.1 mM. Different brain regions such as olfactory bulb (OB), hippocampus (HI), visual cortex (VC), brain stem (BS) and cerebellum (CB) were homogenized in 0.1 M perchloric acid (PCA) containing isoproterenol as an internal standard and the supernatant obtained after centrifugation at 14 000 rev./min, was filtered through a membrane of 0.45/zm pore size (Sartorius, G6ttingen, West Germany). An aliquot of 100 #1 of filtrate was injected into the column. All separations were isocratic at room temperature. Flow rate was set at 0.9 ml/min. Levels of noradrenaline (NA), dopamine (DA) and serotonin (5-HT) after separation were detected at the excitation wavelength of 280 nm and emission wavelength of 315 nm keeping the slit width at 10 for both excitation and emission. Tissue monoamine levels were calculated by comparing peak heights with those of standards and with a correction factor related to the recovery of internal standard.
2.5. Acetylcholinesterase activity Different brain regions were homogenized in phosphate buffer (pH 8.0; 0.1 M) and
M.K. Lakshmana, T.R. Rajul Toxicology 91 (1994) 139-150
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activity was measured by the yellow colour produced in the reaction of thiocholine with dithiobisnitrobenzoate ion (Ellman et al., 1961). The absorbance was measured at 412 nm, using an LKB spectrophotometer. 2.6. Behavioural testing
Endosulfan exposed rats and controls were tested in a Skinner box at age 25 days for operant conditioning with solid food reward on continuous reinforcement (CRF) schedule. Each rat was first exposed to the Skinner box environment in two sessions of 30 min duration on 2 successive days. During these sessions, rats were not given any reward. Rats were then deprived o f food and water for about 24 h, before being placed in the Skinner box. Reward (food pellet) was first given whenever the animal approached the vicinity of the lever and then when the animal sniffed at the lever. Training was accomplished in one or two sessions of 30-min duration. A trained rat pressed the pedal on its own and got the reward at least 30 times over a period of 30 rain. These rats were then tested for the number of pedal presses per 30 rain as a measure of retention of acquired skill. Results were subjected to statistical analysis and the significance of differences between means of control and experimental animals was calculated by using the Students t-test (2-tailed). NORADRENALINE
[•CONTROL
[]
ENDOSULFANTREATED
25 DAYS
350 300 250 E 200 C~
~5o c 100 5O 0
35O 3OO E 250
10 DAYS
200 c 150 tO0 50 0 OB
HI BRAIN
VC
CB
BS
REGIONS
Fig. 2. Changes in the levelsof NA in various brain regions at different age groups. Values are mean and S.D. of six animalsin each group. *P < 0.05, **P < 0.01, control group comparedwith endosulfan group using Student's t-test (2-tailed). OB, olfactory bulb; HI, hippocampus; VC, visual cortex; CB, cerebellum and BS, brainstem
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3. Results 3.1. General observation o f animals Endosulfan-treated rat pups looked normal. No general behavioural abnormalities of any kind were noted in rats during endosulfan consumption. Body and brain weights did not differ from controls (data not included). 3.2. Changes in levels o f monoamines Levels of the following transmitters were compared with those present in the corresponding brain regions of age-matched control rats. As shown in Fig. 2, N A levels were significantly increased in OB (12%; P < 0.01) and in BS (10%; P < 0.05) in endosulfan-exposed rat pups at 10 days of age, while in HI, VC and CB, NA levels were not altered. At 25 days of age, NA levels were significantly increased in HI (20%; P < 0.01) and in CB (12%; P < 0.05); other brain regions like OB, VC and BS did not show any alterations at 25 days of age in endosulfan-exposed rats. Levels of DA shown in Fig. 3 were significantly decreased in OB (27%; P < 0.01) and in HI (42%; P < 0.001) whereas in VC, BS and CB, DA levels were not statistically
DOPAMINE Z] CONTROL ~ ]
ENDOSULFAN TREATED
25 DAYS
300 250 200
S~ 15o c I00 5O 0 300 250 200
10 DAYS
; 1so c
100 50 OB
HI VC BRAIN REGIONS
CB
BS
Fig. 3. Changesin the levels of DA in various brain regions at different age groups. Valuesare mean and S.D. of 6 animals in each group. *P < 0.05, **P < 0.01, ***P < 0.001, control group compared with endosulfan group using Student's t-test (2-tailed). For abbreviations see legend for Fig. 2
M.K. Lakshmana, T.R. Raju / Toxieology 91 (1994) 139-150 5
~
-
145
HYDROXYTRYPTAMINE
CONTROL J~J ENDOSULFAN TREATED
700
25
DAYS
i
600 5OO
I i
~ C
3oo 200 100 0
700 600 E 500
10 DAYS
400 30o
200 I00 0
OB
HI VC CB BRAIN REGIONS
BS
Fig. 4. Changes in the levels of 5-HT in various brain regions at different age groups. Values are mean and S.D. of six animals in each group. *P < 0.05, **P < 0.01, ***P < 0.001, control group compared with endosulfan group using Student's t-test (2-tailed). For abbreviations see legend for Fig. 2
Table 1 Effect of endosulfan (6 mg/kg body weight) on acetylcholine-esterase activity a in various regions of rat brain Brain regions
Age in days 10
Olfactory bulb Hippocampus Visual cortex Brain stem Cerebellum
25
Control
Endosulfan
Control
Endosulfan
1.67 7.81 2.35 9.22 2.16
1.69 7.63 2.17 9.38 2.20
3.72 13.79 5.10 30.41 3.21
3.69 12.89 4.90 28.71 3.57
± 0.07 4- 0.32 4- 0.19 4- 0.59 4- 0.18
± 0.02 4- 0.39 4- 0.09 4- 0.67 4- 0.17
± 0.06 4- 0.63 4- 0.41 4- 0.81 4- 0.32
4- 0.02 4- 0.71 4- 0.51 4- 2.11 4- 0.35
Values represent mean + S.D. of 6 animals in each group, a × 10-6/~mol hydrolysed per min per g tissue. Students' t-test comparison of control group with endosulfan group did not reveal any significant change in all brain regions studied.
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Table 2 Effect of endosulfan (6 mg/kg body weight) on operant learning performance at 25 days of age in continuous reinforcement schedule Test parameter
Control
Endosulfan
Total time (min) for shaping Number of sessions required Number of pedal presses per 30 min
52.61 + 5.5 1.76 4- 0.008 105.30 4- 12.1
67.52 4- 6.31"* 2.25 4- 0.06*** 83.72 4- 8.21**
Values represent mean ± S.D. of 6 animals in each group. **P < 0.01 ***P < 0.001, control group comparedwith experimentalgroup usingstudent's t-test (2-tailed).
different from control at 10 days. However at 25 days of age increased DA levels persisted only in OB (14%; P < 0.05) but in H I the DA level remained significantly low (45%; P < 0.001). DA levels were not altered in VC, BS and CB at 25 days. At 10 days of age, 5-HT levels were significantly increased in OB (12%; P < 0.05), in HI (41%; P < 0.001), in VC (30%; P < 0.01) and in BS (15%; P < 0.01) but no alteration occurred in CB as shown in Fig. 4. In contrast, at 25 days 5-HT levels were decreased in CB (31%; P < 0.01) and in BS (20%; P < 0.05) while in OB, HI and VC, 5-HT levels recovered back to control levels. 3.3. A C h E activity
The activity of AChE in various brain regions of rat pups exposed to endosulfan was not significantly different from control both at 10 and 25 days as shown in Table 1. 3.4. Behavioural test
At 25 days of age, rats which consumed endosulfan took 29% (P < 0.01) more time to learn the task compared to control rats. Even after acquisition, the pedal press rate was lower (21%; P < 0.01) compared to control group of rats (Table 2).
4. Discussion The significant finding of this study is that consumption of endosulfan at low dose for longer duration can ultimately lead to differential alterations of monoamines in various regions of the rat central nervous system. This occurred inspite of the absence of any gross deficits in the body and brain weights. The observed lack of change in the body weight after endosulfan consumption is in support of the earlier observation with comparable dosage (Singh and Pandey, 1989). 4.1. Monoamine alterations - - reconciling with previously published data
In the present study, N A levels were increased in OB and BS at 10 days of age and in HI and CB at 25 days of age. Thus noradrenergic systems responded differently
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to different durations of endosulfan-exposure. In an earlier investigation (Ansari et al., 1987), after i.p. injection of endosulfan at 40 mg/kg body weight in male rats, NA levels were reported to be increased in BS and cerebral cortex. Thus an endosulfaninduced increase in the NA level in BS is confirmed by our study. However we did not observe any alteration in NA level in the visual cortical region. This variance in results might be due to differences in regional susceptibility to different dosages used. Ansari et al. (1987) have reported an increase in DA level only in corpus striatum while in other brain regions endosulfan did not affect DA levels. By contrast, we have observed decreased DA levels in OB and HI at 10 days and at 25 days of age. The decreased DA level persisted only in HI while OB showed an increased DA. Thus endosulfan affected only DA-containing cell bodies (OB, HI) whereas DA-containing terminals (VC, CB) seem to be relatively resistant to the effect of endosulfan. Increased DA level has been reported in the brain of freshwater fish Channa, when exposed to water containing 0.01 ppm endosulfan (Gopal et al., 1985). This variance in result might be due to the acute nature of exposure and whole-brain estimations, unlike the chronic exposure to endosulfan and region specific estimations of monoamines carried out in the present study. In a previous report (Ansari et al., 1987), 5-HT levels were shown to be increased in cerebral cortex and decreased in BS; a decrease in 5-HT level has also been demonstrated in whole-brain estimations (Gopal et al., 1985). We have also observed decreased 5-HT levels in BS but only at 25 days of age whereas at 10 days, levels were increased in OB, HI, VC and BS regions.
4.2. Differential regional vulnerability The data suggest that chronic exposure to endosulfan during the brain growth spurt period of development induced dynamic alterations in the levels of monoamines in various regions studied. Reasons for such selective regional vulnerability are not understood. It may be partly due to differential accumulation of endosulfan in different regions of the rat central nervous system (Gupta, 1978). Local cellular characteristics of neurons and glia may also delineate how endosulfan is metabolized. Changes in transmitter levels observed at 10 days of age are either recovered back to normal control levels at 25 days of age or in some cases beyond control levels. Such dynamic temporal and regional alterations in monoamine levels have been reported after chronic exposure to mercuric chloride (Lakshmana et al., 1993) and another organochlorine pesticide, benzene hexachloride (Nagaraja and Desiraju, in press). Benzene hexachloride exposed from post-natal day 2 decreased NA levels at 20 days of age in cortex and HI (24%; P < 0.01) but DA levels were increased in HI (47%; P < 0.01) and BS (27%; P < 0.01) while 5-HT levels were increased in cortex (23%; P < 0.05) and decreased in HI (27%, P < 0.01) and BS (29%, P < 0.01). Decreased NA levels persisted even at 60 days in cortex and HI (24%; P < 0.01). But DA levels were increased in BS (22%; P < 0.01) and also in CB (39%; P < 0.05) while 5-HT levels returned to control levels in all these regions. On the other hand the organophosphorus pesticide, methyl parathion did not affect the levels of monoamines (Shailesh Kumar and Desiraju, 1992).
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4.3. AChE activity AChE activity was not affected in any of the regions studied in the endosulfantreated rat pups at this dosage. Our finding is consistent with the previous finding that sub-lethal doses of endosulfan do not alter the activity of AChE in whole brains (Inbaraj and Haider, 1988).
4.4. Cognitive deficits Chronic consumption of low dose endosulfan affected the performance of rats in the operant learning paradigm. At 25 days, endosulfan-treated rats took significantly more time for acquisition i.e. the time spent to learn the task was more in experimental rats than in the control group. Even after acquisition, endosulfan-treated rats differed from control rats in retention of the acquired task. The number of pedal presses was significantly lower compared to control group. This is in support of the earlier observation of cognitive deterioration and severe impairment of memory even after 2 years of residual phase in a case of acute poisoning by endosulfan (Aleksandrowicz, 1979). Comparative studies with AChE and cholineacetyl-transferase (CHAT) (Levey et al., 1983) show that the distribution of ChAT immunoreactivity and the AChE-staining pattern are virtually identical. Thus AChE would appear to be a reasonably good indicator for the distribution of cholinergic terminals (Mathews et al., 1987). Since the cholinergic system is implicated in cognitive functions, it is interesting to note that even in the absence of any effect of endosulfan on AChE and ACh levels (Ansari et al., 1987) the rats consuming endosulfan displayed deficits in cognitive functions. This suggests that endosulfan affects cognitive function through non-cholinergic systems, probably monoamines. In fact, monoamines have been implicated in long term potentiation (LTP) in the hippocampus (Stanton and Sarvey, 1985; Frey et al., 1991). Therefore the observed increased NA and decreased DA in HI might be responsible for cognitive deficits in endosulfan-exposed rats.
4.5. Mechanism of action Endosulfan might induce monoamine alterations by directly acting on the enzymes related to their synthesis and degradation. In addition, endosulfan might influence the release and re-uptake of monoamines. The increased NE and 5-HT levels could result from either activation of dopamine ~-hydroxylase and tryptophan hydroxylase, respectively or inhibition of monoamine oxidase. Decreased DA might be due to inhibition of tyrosine hydroxylase. However, our result showing increased NA and decreased DA occurring in the same region (HI) is intriguing and cannot be explained on this basis. A possible effect of endosulfan on specific enzymes related to monoamine metabolism is currently being investigated in our laboratory. 5. References Aleksandrowicz, D.R. (1979) Endosulfan poisoning and chronic brain syndrome. Arch. Toxicol. 43, 65.
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Nagaraja, T.N. and Desiraju,T. Brain regional variations in the levels of biogenic amines, glutamate, GABA and glutamate decarboxylase activity in developing and adult rats exposed chronically to hexachlorocyclohexane. Biogenic Amines, In press. Shailesh Kumar, M.V. and Desiraju, T. (1992) Effect of chronic consumption of methylparathion on rat brain regional acetylcholinesterase activity and on levels of biogenic amines. Toxicology 75, 262. Singh, S.K. and Pandey, R.S. (1989) Differential effects of chronic endosulfan exposure to male rats in relation to hepatic drug metabolism and androgen biotransformation. Indian J. Biochem. Biophys. 26, 262. Stanton, P.K. and Sarvey, J.M. (1985) Depletion of norepinephrine, but not serotonin reduces long term potentiation in the dentate gyrus of rat hippocampal slices. J. Neurosci. 5, 2169. Strachan, W.M.J., Huneault, H., Schertzer, W.M. and Elder, F.C. (1980) Organo chlorines in precipitation in the Great Lakes region. In: B.K. Afghan and D. McKay (Eds), Hydrocarbons and Halogenated Hydrocarbons in the Aquatic Environment, Plenum Press, New York, p. 387. Terziev, G., Dimitrova, N. and Rusev, P. (1974) Forensic medical and forensic chemcial study of acute lethal poisonings with thiodan. Folia Med. 16, 325. Tiberin, P., Kristal, N. and Israeli, R. (1970) EEG findings in poisoning by endosulfan. Electroencephalogr. Clin. Neurophysiol. 28, 642. WHO report, Environmental Health Criteria, 40, World Health Organization, Geneva, 1984. Zaidi, N.F., Agarwal, A.K., Anand, M. and Seth, P.K. (1985) Neo-natal endosulfan neurotoxicity, behavioral and biochemical changes in rat pups. Neurobehav. Toxicol. Teratol. 7, 439.
Toxicology 91 (1994) 139-150
Endosulfan induces small but significant changes in the levels of noradrenaline, dopamine and serotonin in the developing rat brain and deficits in the operant learning performance Madepalli K. Lakshmana, Trichur R. Raju* Department of Neurophysiology, National Institute of Mental Health and Neuro Sciences, Bangalore-560 029, India
(Received 18 August 1993; accepted 21 November 1993)
Abstract
The organochlorine insecticide, endosulfan was administered (6 mg/kg body weight) to Wistar rat pups of both sexes by gastric intubation daily from post-natal days 2-25. Its effect on levels of noradrenaline (NA), dopamine (DA) and serotonin (5-HT) was assayed in olfactory bulb (OB), hippocampus (HI), visual cortex (VC), brainstem (BS) and cerebellum (CB) on days 10 and 25 using high-performance liquid chromatography (HPLC). The activity of acetylcholinesterase (ACHE) was also estimated in the same regions of the brain. Performance in operant conditioning for solid food reward was assessed in 25-day-old rats. NA levels were increased in OB (12%, P < 0.01) and BS (10%, P < 0.05) at 10 days of age and in HI (20%, P < 0.01) and CB (12%, P < 0.05) at 25 days of age. DA level was decreased in H1 at both 10 (42%, P < 0.001) and 25 (45%, P < 0.001) days. Serotonin levels were increased in OB (12%, P < 0.05), HI (41%, P < 0.001), VC (30%, P < 0.01) and BS (15%, P < 0.01) at 10 days of age but at 25 days, levels were decreased in BS (20%, P < 0.05) and CB (31%, P < 0.01). The activity of AChE was not different from the control groups in any of the regions studied. These data suggest that monoaminergic systems in the developing rat brain respond to endosulfan by undergoing something like a 'reorganization'. However, such changes do not ameliorate certain functional losses following the exposure to endosulfan as operant conditioning revealed deficits in acquisition as well as retention of memory. Abbreviations: ACHE, Aeetylcholine esterase; BS, Brainstem; CB, Cerebellum; DA, Dopamine; HI, Hippoeampus; 5-HT, 5-hydroxytryptamine; NA, Noradrenaline; OB, Olfactory bulb; VC, Visual cortex * Corresponding author.
0300-483X/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0300-483X(93)02772-Z
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Keywords:Chronic endosulfan exposure; Developing rat brain; Monoamines; ACHE; Operant conditioning; HPLC
1. Introduction
Endosulfan, an organochlorine insecticide of cyclodiene structure has substantially replaced the earlier persistent type of chlorinated compounds such as dichlorodiphenyltrichloro acetic acid (DDT) and benzene hexachloride (BHC) and is now much in use for agriculture and crop protection (WHO, 1984). Endosulfan has been detected in ambient air samples (Kutz et al., 1976), in precipitation in the Great Lakes (Strachan et al., 1980) and in cow's milk (Frank et al., 1979) though at lower concentrations. High levels of endosulfan residues have also been reported in tobacco smoke (Chopra et al., 1978; Cheng and Braun, 1977). Clinical signs of endosulfan toxicity in accidental poisoning (Terziev et al., 1974) and in workers employed in chemical factories (Tiberin et al., 1970), suggest the involvement of the central nervous system and include symptoms such as hyperactivity, irritability, disorientation, tremors and convulsions followed by death. Toxicity of endosulfan to experimental animals has been reported (Gupta and Gupta, 1979). Altered electrical activity of the brain has been found after endosulfan administration, in cats (Anand, 1980) and in rats (Anand et al., 1980). However, the mechanism by which endosulfan induces toxicity remains elusive. Zaidi et al. (1985) have implicated the serotonergic system in endosulfan neurotoxicity in neo-natal rats from binding studies using 5-hydroxytryptamine (5-HT) blocker, methysergide; this was correlated with behavioural changes. Chronic endosulfan exposure has been shown to inhibit 5-HT uptake (Anand et al., 1986a) which supports the above finding. On the other hand Anand et al. (1986b) have suggested the involvement of the cholinergic system in the mediation of endosulfan neurotoxicity. It is possible that endosulfan may mediate its neurotoxic effect by acting on different neurotransmitter systems influencing their synthesis, release, uptake and degradation. Ansari et al. (1987) studied the effect of acute endosulfan exposure on different neurotransmitters of brain after a single intraperitoneal (i.p.) dose in male rats. They showed alterations in the levels of noradrenaline (NA), dopamine (DA) and 5-HT but not acetylcholine (ACh). However, a very high dose, about half of the lethal dose, was used in this study. Since endosulfan exposure is mainly through food intake, at relatively lower concentrations, it is not known whether chronic consumption of endosulfan at lower doses for longer duration could have similar effects. The possible effect of endosulfan on the developing nervous system, which is more vulnerable to external insults than the adult brain is also not yet studied. Accordingly, the present study was designed to investigate the effect of endosulfan consumption in a lower dose during post-natal brain growth period on the levels of NA, DA and 5-HT and on the activity of acetylcholinesterase (ACHE). As the accumulation of endosulfan has been shown to be uneven in the central nervous system (Gupta, 1978) these parameters were estimated in various regions of developing rat brain. In addition, rats were also subjected to operant conditioning, a paradigm used for testing learning and memory.
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2. Materials and methods
2.1. Materials
Endosulfan (6,7,8,9,10,10-hexachloro- 1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3benzodioxa-thiepin-3-oxide) obtained from Excel Co., Bombay, India, was of technical grade (96%). Standards such as norepinephrine bitartrate salt, dopamine hydrochloride, 5-hydroxytryptamine hydrochloride, isoproterenol hydrochloride and heptane sulfonic acid for monoamine estimations and acetylthiocholine iodide, dithiobisnitrobenzoate (DTNB) for AChE assay were all obtained from Sigma Chemical Co., St. Louis, MO, USA. Methanol of HPLC grade was obtained from Spectrochemicals, Bombay, India. Ethylenediaminetetraacetic acid (EDTA), sodium acetate, sodium bicarbonate, orthophosphoric acid and perchloric acid (70%) of analytical grade were all purchased from a local supplier, BDH, Bombay, India. Doubledistilled water was used for preparing all solutions. 2.2. Animals
Female Wistar rats were caged individually after confirmation of conception. Litter size was maintained at eight pups per mother. Rat pups of both sexes were allowed to be with mother throughout the day during weaning and thereafter had ad libitum access to water and a semi-synthetic balanced diet (18% casein, 78% wheat) (Mascarenhas et al., 1984) which contains all vitamins and minerals for the normal growth of rats. The room temperature was 25-26°C and 12- h light and dark cycles were strictly enforced in a fully ventilated room. 2.3. Experimental procedure
As shown in Fig. 1, the preliminary experiments in our laboratory revealed 8% inhibition of Na +, K + ATPase activity in rat brain microsomes as determined by the method of Davis (1970) at 10-6 M endosulfan. The presently observed Na+,K + ATPase inhibition was small at 10-6M (equivalent to 4.1 mg/kg body weight). Also, in a previous study, since no toxic symptoms were observed in rats at <__5 mg/kg body weight of endosulfan by oral intubation (Garg et al., 1980), in the present study 1.5 times this dosage was used. A group of experimental rats, were fed endosulfan (6 mg/kg body weight) (dissolved in peanut oil) daily between 09.00-10.00 h. Control groups of rat pups received equal amounts of peanut oil alone by the same route. Rat pups from control and experimental groups were sacrificed by decapitation on day 10 (n = 6 in each group) and day 25 (n = 6 in each group). Brains were removed immediately and different brain regions were dissected on an ice-cold Petri dish as described by Lindgren et al. (1982). 2.4. Monoamine estimations
The HPLC system consisted of a delivery pump (model ERC-8710, Erma Optical Works, Tokyo, Japan), a reverse-phase analytical column, Ultracarb 3 /~m ODS,
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M.K. Lakshmana, T.R. Raju / Toxicology 91 (1994) 139-150 100 O_
~- 80 i
+x-" ÷4
z
60
C
0
~_- 40 e-
~ 20 n i
166 Endosulfan
i
10-5
i
10-4
Concentration ( M )
Fig. 1. Inhibition of Na+K+-ATPase by endosulfan in rat brain microsomes. The ATPase activity was assayed by the liberation of Pi from the substrate ATP in 15 min at pH 7.5 and 35°C. Inhibition is expressed relative to a solvent control. Each point represents the mean ( ± S.E.) of six independent experiments performed on different days with brain microsomes.
150 × 4.6 mm (Phenomenex, Torrance, CA, USA), a guard column, Ultracarb 3 t~m ODS, 30 x 4.6 mm (phenomenex, Torrance, CA, USA), a degasser (model ERC 3310, ERMA INC, Tokyo, Japan), a recorder (Sekonic SS 100F) and a fluorescence spectrophotometer (Hitachi, model 650-40, Japan) for detection. Samples were prepared and analysed by a slightly modified procedure of Murai et al. (1988). The modified mobile phase, pH 3.92, consisted of sodium acetate 0.02 M, methanol 16% (v/v), heptane sulfonic acid 0.1375% (w/v) and EDTA 0.1 mM. Different brain regions such as olfactory bulb (OB), hippocampus (HI), visual cortex (VC), brain stem (BS) and cerebellum (CB) were homogenized in 0.1 M perchloric acid (PCA) containing isoproterenol as an internal standard and the supernatant obtained after centrifugation at 14 000 rev./min, was filtered through a membrane of 0.45/zm pore size (Sartorius, G6ttingen, West Germany). An aliquot of 100 #1 of filtrate was injected into the column. All separations were isocratic at room temperature. Flow rate was set at 0.9 ml/min. Levels of noradrenaline (NA), dopamine (DA) and serotonin (5-HT) after separation were detected at the excitation wavelength of 280 nm and emission wavelength of 315 nm keeping the slit width at 10 for both excitation and emission. Tissue monoamine levels were calculated by comparing peak heights with those of standards and with a correction factor related to the recovery of internal standard.
2.5. Acetylcholinesterase activity Different brain regions were homogenized in phosphate buffer (pH 8.0; 0.1 M) and
M.K. Lakshmana, T.R. Rajul Toxicology 91 (1994) 139-150
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activity was measured by the yellow colour produced in the reaction of thiocholine with dithiobisnitrobenzoate ion (Ellman et al., 1961). The absorbance was measured at 412 nm, using an LKB spectrophotometer. 2.6. Behavioural testing
Endosulfan exposed rats and controls were tested in a Skinner box at age 25 days for operant conditioning with solid food reward on continuous reinforcement (CRF) schedule. Each rat was first exposed to the Skinner box environment in two sessions of 30 min duration on 2 successive days. During these sessions, rats were not given any reward. Rats were then deprived o f food and water for about 24 h, before being placed in the Skinner box. Reward (food pellet) was first given whenever the animal approached the vicinity of the lever and then when the animal sniffed at the lever. Training was accomplished in one or two sessions of 30-min duration. A trained rat pressed the pedal on its own and got the reward at least 30 times over a period of 30 rain. These rats were then tested for the number of pedal presses per 30 rain as a measure of retention of acquired skill. Results were subjected to statistical analysis and the significance of differences between means of control and experimental animals was calculated by using the Students t-test (2-tailed). NORADRENALINE
[•CONTROL
[]
ENDOSULFANTREATED
25 DAYS
350 300 250 E 200 C~
~5o c 100 5O 0
35O 3OO E 250
10 DAYS
200 c 150 tO0 50 0 OB
HI BRAIN
VC
CB
BS
REGIONS
Fig. 2. Changes in the levelsof NA in various brain regions at different age groups. Values are mean and S.D. of six animalsin each group. *P < 0.05, **P < 0.01, control group comparedwith endosulfan group using Student's t-test (2-tailed). OB, olfactory bulb; HI, hippocampus; VC, visual cortex; CB, cerebellum and BS, brainstem
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3. Results 3.1. General observation o f animals Endosulfan-treated rat pups looked normal. No general behavioural abnormalities of any kind were noted in rats during endosulfan consumption. Body and brain weights did not differ from controls (data not included). 3.2. Changes in levels o f monoamines Levels of the following transmitters were compared with those present in the corresponding brain regions of age-matched control rats. As shown in Fig. 2, N A levels were significantly increased in OB (12%; P < 0.01) and in BS (10%; P < 0.05) in endosulfan-exposed rat pups at 10 days of age, while in HI, VC and CB, NA levels were not altered. At 25 days of age, NA levels were significantly increased in HI (20%; P < 0.01) and in CB (12%; P < 0.05); other brain regions like OB, VC and BS did not show any alterations at 25 days of age in endosulfan-exposed rats. Levels of DA shown in Fig. 3 were significantly decreased in OB (27%; P < 0.01) and in HI (42%; P < 0.001) whereas in VC, BS and CB, DA levels were not statistically
DOPAMINE Z] CONTROL ~ ]
ENDOSULFAN TREATED
25 DAYS
300 250 200
S~ 15o c I00 5O 0 300 250 200
10 DAYS
; 1so c
100 50 OB
HI VC BRAIN REGIONS
CB
BS
Fig. 3. Changesin the levels of DA in various brain regions at different age groups. Valuesare mean and S.D. of 6 animals in each group. *P < 0.05, **P < 0.01, ***P < 0.001, control group compared with endosulfan group using Student's t-test (2-tailed). For abbreviations see legend for Fig. 2
M.K. Lakshmana, T.R. Raju / Toxieology 91 (1994) 139-150 5
~
-
145
HYDROXYTRYPTAMINE
CONTROL J~J ENDOSULFAN TREATED
700
25
DAYS
i
600 5OO
I i
~ C
3oo 200 100 0
700 600 E 500
10 DAYS
400 30o
200 I00 0
OB
HI VC CB BRAIN REGIONS
BS
Fig. 4. Changes in the levels of 5-HT in various brain regions at different age groups. Values are mean and S.D. of six animals in each group. *P < 0.05, **P < 0.01, ***P < 0.001, control group compared with endosulfan group using Student's t-test (2-tailed). For abbreviations see legend for Fig. 2
Table 1 Effect of endosulfan (6 mg/kg body weight) on acetylcholine-esterase activity a in various regions of rat brain Brain regions
Age in days 10
Olfactory bulb Hippocampus Visual cortex Brain stem Cerebellum
25
Control
Endosulfan
Control
Endosulfan
1.67 7.81 2.35 9.22 2.16
1.69 7.63 2.17 9.38 2.20
3.72 13.79 5.10 30.41 3.21
3.69 12.89 4.90 28.71 3.57
± 0.07 4- 0.32 4- 0.19 4- 0.59 4- 0.18
± 0.02 4- 0.39 4- 0.09 4- 0.67 4- 0.17
± 0.06 4- 0.63 4- 0.41 4- 0.81 4- 0.32
4- 0.02 4- 0.71 4- 0.51 4- 2.11 4- 0.35
Values represent mean + S.D. of 6 animals in each group, a × 10-6/~mol hydrolysed per min per g tissue. Students' t-test comparison of control group with endosulfan group did not reveal any significant change in all brain regions studied.
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Table 2 Effect of endosulfan (6 mg/kg body weight) on operant learning performance at 25 days of age in continuous reinforcement schedule Test parameter
Control
Endosulfan
Total time (min) for shaping Number of sessions required Number of pedal presses per 30 min
52.61 + 5.5 1.76 4- 0.008 105.30 4- 12.1
67.52 4- 6.31"* 2.25 4- 0.06*** 83.72 4- 8.21**
Values represent mean ± S.D. of 6 animals in each group. **P < 0.01 ***P < 0.001, control group comparedwith experimentalgroup usingstudent's t-test (2-tailed).
different from control at 10 days. However at 25 days of age increased DA levels persisted only in OB (14%; P < 0.05) but in H I the DA level remained significantly low (45%; P < 0.001). DA levels were not altered in VC, BS and CB at 25 days. At 10 days of age, 5-HT levels were significantly increased in OB (12%; P < 0.05), in HI (41%; P < 0.001), in VC (30%; P < 0.01) and in BS (15%; P < 0.01) but no alteration occurred in CB as shown in Fig. 4. In contrast, at 25 days 5-HT levels were decreased in CB (31%; P < 0.01) and in BS (20%; P < 0.05) while in OB, HI and VC, 5-HT levels recovered back to control levels. 3.3. A C h E activity
The activity of AChE in various brain regions of rat pups exposed to endosulfan was not significantly different from control both at 10 and 25 days as shown in Table 1. 3.4. Behavioural test
At 25 days of age, rats which consumed endosulfan took 29% (P < 0.01) more time to learn the task compared to control rats. Even after acquisition, the pedal press rate was lower (21%; P < 0.01) compared to control group of rats (Table 2).
4. Discussion The significant finding of this study is that consumption of endosulfan at low dose for longer duration can ultimately lead to differential alterations of monoamines in various regions of the rat central nervous system. This occurred inspite of the absence of any gross deficits in the body and brain weights. The observed lack of change in the body weight after endosulfan consumption is in support of the earlier observation with comparable dosage (Singh and Pandey, 1989). 4.1. Monoamine alterations - - reconciling with previously published data
In the present study, N A levels were increased in OB and BS at 10 days of age and in HI and CB at 25 days of age. Thus noradrenergic systems responded differently
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to different durations of endosulfan-exposure. In an earlier investigation (Ansari et al., 1987), after i.p. injection of endosulfan at 40 mg/kg body weight in male rats, NA levels were reported to be increased in BS and cerebral cortex. Thus an endosulfaninduced increase in the NA level in BS is confirmed by our study. However we did not observe any alteration in NA level in the visual cortical region. This variance in results might be due to differences in regional susceptibility to different dosages used. Ansari et al. (1987) have reported an increase in DA level only in corpus striatum while in other brain regions endosulfan did not affect DA levels. By contrast, we have observed decreased DA levels in OB and HI at 10 days and at 25 days of age. The decreased DA level persisted only in HI while OB showed an increased DA. Thus endosulfan affected only DA-containing cell bodies (OB, HI) whereas DA-containing terminals (VC, CB) seem to be relatively resistant to the effect of endosulfan. Increased DA level has been reported in the brain of freshwater fish Channa, when exposed to water containing 0.01 ppm endosulfan (Gopal et al., 1985). This variance in result might be due to the acute nature of exposure and whole-brain estimations, unlike the chronic exposure to endosulfan and region specific estimations of monoamines carried out in the present study. In a previous report (Ansari et al., 1987), 5-HT levels were shown to be increased in cerebral cortex and decreased in BS; a decrease in 5-HT level has also been demonstrated in whole-brain estimations (Gopal et al., 1985). We have also observed decreased 5-HT levels in BS but only at 25 days of age whereas at 10 days, levels were increased in OB, HI, VC and BS regions.
4.2. Differential regional vulnerability The data suggest that chronic exposure to endosulfan during the brain growth spurt period of development induced dynamic alterations in the levels of monoamines in various regions studied. Reasons for such selective regional vulnerability are not understood. It may be partly due to differential accumulation of endosulfan in different regions of the rat central nervous system (Gupta, 1978). Local cellular characteristics of neurons and glia may also delineate how endosulfan is metabolized. Changes in transmitter levels observed at 10 days of age are either recovered back to normal control levels at 25 days of age or in some cases beyond control levels. Such dynamic temporal and regional alterations in monoamine levels have been reported after chronic exposure to mercuric chloride (Lakshmana et al., 1993) and another organochlorine pesticide, benzene hexachloride (Nagaraja and Desiraju, in press). Benzene hexachloride exposed from post-natal day 2 decreased NA levels at 20 days of age in cortex and HI (24%; P < 0.01) but DA levels were increased in HI (47%; P < 0.01) and BS (27%; P < 0.01) while 5-HT levels were increased in cortex (23%; P < 0.05) and decreased in HI (27%, P < 0.01) and BS (29%, P < 0.01). Decreased NA levels persisted even at 60 days in cortex and HI (24%; P < 0.01). But DA levels were increased in BS (22%; P < 0.01) and also in CB (39%; P < 0.05) while 5-HT levels returned to control levels in all these regions. On the other hand the organophosphorus pesticide, methyl parathion did not affect the levels of monoamines (Shailesh Kumar and Desiraju, 1992).
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4.3. AChE activity AChE activity was not affected in any of the regions studied in the endosulfantreated rat pups at this dosage. Our finding is consistent with the previous finding that sub-lethal doses of endosulfan do not alter the activity of AChE in whole brains (Inbaraj and Haider, 1988).
4.4. Cognitive deficits Chronic consumption of low dose endosulfan affected the performance of rats in the operant learning paradigm. At 25 days, endosulfan-treated rats took significantly more time for acquisition i.e. the time spent to learn the task was more in experimental rats than in the control group. Even after acquisition, endosulfan-treated rats differed from control rats in retention of the acquired task. The number of pedal presses was significantly lower compared to control group. This is in support of the earlier observation of cognitive deterioration and severe impairment of memory even after 2 years of residual phase in a case of acute poisoning by endosulfan (Aleksandrowicz, 1979). Comparative studies with AChE and cholineacetyl-transferase (CHAT) (Levey et al., 1983) show that the distribution of ChAT immunoreactivity and the AChE-staining pattern are virtually identical. Thus AChE would appear to be a reasonably good indicator for the distribution of cholinergic terminals (Mathews et al., 1987). Since the cholinergic system is implicated in cognitive functions, it is interesting to note that even in the absence of any effect of endosulfan on AChE and ACh levels (Ansari et al., 1987) the rats consuming endosulfan displayed deficits in cognitive functions. This suggests that endosulfan affects cognitive function through non-cholinergic systems, probably monoamines. In fact, monoamines have been implicated in long term potentiation (LTP) in the hippocampus (Stanton and Sarvey, 1985; Frey et al., 1991). Therefore the observed increased NA and decreased DA in HI might be responsible for cognitive deficits in endosulfan-exposed rats.
4.5. Mechanism of action Endosulfan might induce monoamine alterations by directly acting on the enzymes related to their synthesis and degradation. In addition, endosulfan might influence the release and re-uptake of monoamines. The increased NE and 5-HT levels could result from either activation of dopamine ~-hydroxylase and tryptophan hydroxylase, respectively or inhibition of monoamine oxidase. Decreased DA might be due to inhibition of tyrosine hydroxylase. However, our result showing increased NA and decreased DA occurring in the same region (HI) is intriguing and cannot be explained on this basis. A possible effect of endosulfan on specific enzymes related to monoamine metabolism is currently being investigated in our laboratory. 5. References Aleksandrowicz, D.R. (1979) Endosulfan poisoning and chronic brain syndrome. Arch. Toxicol. 43, 65.
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