Effects of two pyrethroids, bioallethrin and deltamethrin, on subpopulations of muscarinic and nicotinic receptors in the neonatal mouse brain

~ONI(‘OLOGYANUAPPL~~UPHARMAC‘OI.OGY 102,456-463(1990)

Effects of Two Pyrethroids, Bioallethrin and Deltamethrin, on Subpopulations of Muscarinic and Nicotinic

Receptors in the Neonatal Mouse Brain

PER ERIKSSON* AND AGNETA NORDBERG~

Effects ofTwo Pyrethroids, Bioallethrin and Deltamethrin. on Subpopulations of Muscarinic and Nicotinic Receptors in the Neonatal Mouse Brain. ERIKSSON. P.. AND NORDBERG, A. ( 1990). To~-ico/ .Appl. Phurtnuc~o/. 102, 456-463. Ten-day-old NMRI mice were given deltamethrin. bioallethrin. or the vehicle once daily for 7 days. The doses used were as follows: deltamethrin. 0.7 I and I .L! mg/kg body wt; bioallethrin. 0.72 and 72 mg/kg body wt: and ?O’% fat emulsion vehicle. IO ml/kg body wt. The mice were killed 24 hr after the last administration. and crude synaptosomal fractions (P?) were prepared from the cerebral cortex and hippocampus. The densities ofthe muscarinic and nicotinic receptors were assayed by measuring the amounts of quinuclidinyl benzilate ([‘HIQNB) and [“HInicotine, respectively. specifically bound in the PZ fraction. The proportions of high- and low-affinity binding sites of the muscarinic receptors were assayed in a displacement study using [3H]QNB/carbachol. The two types of pyrethroids affected the cholinergic system in the neonatal mouse brain in two different ways. At the lower dose, which did not cause any neurotoxic symptoms, both pyrethroid types affected the muscarinic receptors in the cerebral cortex. Here deltamethrin caused an increase and decrease in the percentage of high- and low-affinity binding sites, respectively, whereas the reverse was observed after bioallethrin treatment. Deltamethrin treatment also caused an increase in the density of nicotinic receptors in the cerebral cortex. The higher doses revealed typical symptoms of pyrethroid poisoning. such as choreoathetosis and tremor for deltamethrin and bioallethrin. respectively. The symptoms declined gradually during each successive day of administration and had disappeared by Day 4. 4t this dose deltamethrin affected the muscarinic receptors in the hippocampus and the nicotinic receptors in the cerebral cortex. whereas bioallethrin had no apparent effect. This study further supports that the cholinergic system under rapid development in the neonatal mouse is sensitive to xenobiolics. 11) 1990 Acadcmao Press. Inc.

The pyrethroids are derivatives of natural py- ofexposure to the former is tremor while cho- rethrins (Elliott et al., 1967; Elliott, 1977). reoathetosis and salivation are the main Due to their highly insecticidal properties py- symptoms of the latter. Most of the toxicolog- rethroids have achieved widespread agricul- ical and physiological investigations concern- tural and environmental health applications. ing the effects of pyrethroids have been per- Pyrethroids are commonly divided into Type formed on adult animals receiving high doses I compounds (or T-syndrome pyrethroids), and on in vitro systems using nerve mem- which lack an cY-cyano substituent, and Type brane preparations (for reference see Casida II compounds (or CS-syndrome pyrethroids), et al., 1983; Gray and Soderlund, 1985; Nara- which contain an a-cyanophenoxybenzyl hashi, 1985). The results from invertebrate substituent (Gammon et al.. 198 1; Versch- and nonmammalian vertebrate preparations oyle and Ahidge, 1980). The main symptom indicate that the responses seen in mamma-

004 I-008X/90 $3.00 Copyright Q 1990 by Academic Press, Inc. All rights of reproduction in any form reserved

456

PYRETHROIDS-NEONATAL NEUROTOXICITY 457

lian studies are qualitatively similar. Obser- vations made on nonmammalian nerve membrane preparations show that one of the basic mechanisms of pyrethroid action in- volves interference with the nerve membrane sodium channels, leading to prolonged depo- larization and induction of repetitive activity (Wouters and van den Bercken, 1978; Lund and Narahashi, 1983; Vijverberg and van den Bercken, 1982; Narahashi 1985). Effects on sodium channels have also been observed in the mouse and rat brain (Ghiasuddin and Soderlund, 1985: Brown et al., 1988) and in mammalian neuroblastoma cells (Roche et al.. 1985).

Despite these well-documented neurotoxic effects of pyrethroids in mammalian and nonmammalian species little is known about the effects in immature animals and the effects of doses to low to cause symptoms. In previous investigations we found that DDT, which shows similarities to pyrethroids, both with respect to neurotoxic symptoms and neurotoxic action (Woolley, 1982; Nara- hashi, 1982) at low doses affects the cholinergic receptors in both immature and adult mice (Eriksson et al.. 1984; Eriksson and Nordberg, 1986). There are also indications that the pyrethroid poisoning in mammals may involve the cholinergic system. A decrease in brain acetylcholine (ACh) concentrations has been observed in rats exposed to deltamethrin (Alridge et al.. 1978). Effects on the rat central nervous system (CNS) observed after deltamethrin exposure include changes in EEG pattern and glucose concentration, the latter of which can effect the ACh concentration (Ray, 1980; Cremer et al., 1980). Although pyrethroids show low acute toxicity and are rapidly metabolized in mammals they might be transferred from mother to offspring via the milk (Kavlock et a/., 1979), and whenever reaching the nervous system in sufficient concentrations they may cause adverse neurotoxic effects.

Therefore, the present investigation was undertaken to examine whether pyrethroids affect the muscarinic and/or nicotinic recep-

tors in the immature mouse brain. Are there any similarities to the effects observed after DDT exposure? Are the changes induced in animals showing neurotoxic symptoms different from those induced in asymptom- atic animals? Do Type I and Type II pyrethroids differ with respect to their effects on receptors?

METHODS

Ten-day-old NMRI mice of both sexes. which were offspring of pregnant mice purchased from ALAB. Swe- den, were used. Each litter. adjusted within 24 hr to 8- 10 mice, was kept together with its respective mother in a plastic cage in a room at a temperature of 22°C and a 12/12-hr light/dark cycle. The animals were supplied with standardized pellet food (Ewes. Sweden) and tap water ad libittrtn.

Deltamethrin (,S-n-cyano-3-phenoxybenzyl-( IR)-cis- 3-(2,2-dibromovinyl)-2.2-dimethylcyclopropane carboxylate) (mw 505) and bioallethrin (2-allyl-4-hydroxy- 3-methyl-2-cyclopenten- 1 -one-( I R,3R,4S)-2.2-dimethylcyclopropane carboxylate) (mw 302) were generous gifts from Dr. Tessier (Roussell Uclaf. France). The substances were dissolved in a mixture of egg lecithin (Merck) and peanut oil (Oleum arachidis) (1:lO. w:w) and were thereafter sonicated together with water to yield a 20% (w:w) fat emulsion vehicle containing deltamethrin at concentrations of 0.07 I and 0.12 mg/ml or bioallethrin at concentrations of 0.072 and 7.2 mg/ml. The substances were administered orally via a PVC tube (di- ameter 1 .O mm) as one single dose per day for 7 days in order to simulate the exposure time effective for DDT (Eriksson and Nordberg. 1986). The amounts of pyrethroids given were as follows; deltamethrin, 0.7 1 mg (1.4 wmol) and 1.2 mg (2.4 rmol)/kg body wt: bioallethtin. 0.72 mg (2.4 pmol) and 72 mg (0.24 mmol)/kg body wt. The deltamethrin dose of 1.4 pmol/kg body wt corresponds (on a molar basis) to the amount of DDT given in an earlier experiment (see Eriksson and Nordberg, 1986). This administration will also give about an equal amount ofthese two insecticides in the neonatal mouse brain (Er- iksson and Darnerud. 1985: Eriksson and Damerud. un- published observation regarding the retention of deltamethrin in the neonatal mouse brain). The bioallethrin dose of 0.72 mg (2.4 pmol)/kg body wt corresponds (on a molar basis) to the deltamethrin dose of 1.2 mg (2.4 pmol)/kg body wt. which causes the neurotoxic symptom choreoathetosis in lo-day-old mice. Mice serving as controls received 10 ml/kg body wt of the 20% fat emulsion vehicle in the same manner.

The mice were killed by decapitation 24 hr after the last treatment. and a crude synaptosomal P2 fraction

458 ERIKSSON AND NORDBERG

(Gray and Whittaker, 1962) from the cerebral cortex and hippocampus with a protein content of about 2 mg/ml (measured according to Lowry et al.. 195 1) was prepared as earlier described (Eriksson and Nordberg. 1986). The P2 fractions were kept frozen (-25°C) until assayed (within 4 months).

Measurements of the muscarinic receptor density and proportion of muscarinic high- and low-affinity binding sites were performed following the methods of Nordberg and Winblad (1981) and Nordberg and Wahlstriim (1982). respectively, and earlier described by Eriksson and Nordberg (I 986). Briefly. the assays were performed by measuring tritium-labeled quinuclidinyl benzilate (13H]QNB; sp act I .44 TBq/mmol. 0.2 and 0.1 IIM, respectively, Amersham International plc, UK) specifically bound in the P2 fraction by using atropin ( 10m4 M) for measuring the nonspecific binding and different concentrations of carbachol (lO~‘-lO~~’ M) for measuring the proportions of high- and low-affinity binding sites. respectively. The specific binding constituted about 98%~ of the total [3H]QNB binding. In analyzing the proportions of the high- and low-affinity binding sites, the data of the competitive displacement of [‘H]QNB by carbachol were fitted by a nonlinear least-squares method as described by Birdsall et ul. (I 978). The computerized model in Graph Pad was used to calculate the percentage of high- and low-affinity binding sites with corresponding affinity constants.

Measurement of the high-affinity nicotinic binding sites was performed by using the tritium-labeled nicotine (S[M+3H]nicotine. sp act 3. I GBq/rmol. ISOLAB, Up- psala, Sweden) following the method of Zhang 1’1 ui. ( 1987). An aliquot of the P2 fraction (120 $) was incu- bated with t3H]nicotine (100 ~1, 5 nM) for 40 min at 4°C in 50 IIIM Tris-HCI buffer (pH 8.0) in a total volume of I .O ml. In parallel samples nicotine (100 ~1, IO ‘M) was present for measuring the nonspecific binding. Each

binding was determined in duplicate. The incubation was terminated by rapid filtration over a Whatman GF/C filter treated with 0.05% polyethyleneimine to eliminate displaceable filter binding. The filters were washed twice with 2-ml portions of ice-cold buffer. After washing. the filters were dried and placed into miniscintillation vials. A 5-ml amount of Quickzint 2000 (Zinsser Analytic. Ltd., UK) liquid scintillation fluid was added to the vials,

and the radioactivity was counted in a scintillation an- alyzer (Packard Tri-Carb 1900 CA). The specitic binding was determined as the difference of the amount bound in the presence and in the absence of nicotine and constituted about 73% of the total [‘HInicotine binding. The counting efficiency was about 42%. and the quench was corrected by using the external standard method.

The Student t test was used in evaluating diflerences between controls and treated animals except for the evaluation of the affinity constants. where the Mann-Whit- ney (! test was used.

RESULTS

Mice receiving the highest dose of deltamethrin ( 1.2 mg/kg body wt) developed choreoathetosis within 1 hr after administration. The symptoms persisted for about 5 hr on the first day and 3 hr on the second, while on the third day of administration only slight symptoms were observed in some mice. From the fourth day of administration until the day of killing no neurotoxic symptoms were observed. Mice administered the lower dose of deltamethrin (0.71 mg/kg body wt) were symptom-free throughout the experiment. The temporary pattern of symptom expres- sion in mice administered the highest dose of bioallethrin (72 mg/kg body wt) was about the same as that observed in mice receiving the highest dose of deltamethrin. Within 1 hr of administering bioallethrin, the mice developed a whole-body tremor, which lasted for about 3 hr. On the second day the symptoms persisted for about 1 hr, and on the third day only slight symptoms were observed. Mice receiving the lower dose of bioallethrin (0.72 mg/kg body wt) did not show any symptoms.

The repeated daily administration ofdelta- methrin to IO-day-old mice for 7 days affected the muscarinic and nicotinic receptors in the immature mouse brain, whereas the corresponding treatment with bioallethrin only affected the muscarinic receptors. Both deltamethrin (0.7 1 mg/kg body wt) and bioallethrin (0.72 mg/kg body wt) significantly increased the amount of specific [“HJ- QNB binding sites in the cerebral cortex (Ta- ble 1). At this dose, however, no significant change was seen in the hippocampus. At the higher doses, which caused neurotoxic symptoms, no significant change in the specific [3H]QNB binding sites in the cerebral cortex was noted. However, deltamethrin (1.2 mg/ kg body wt) caused a significant decrease in the specific [3H]QNB binding sites in the hippocampus. Deltamethrin, at both doses used, significantly increased the amount of the specific [‘HInicotine binding sites in the cerebral cortex (Table 2). In contrast, no significant


TABLE I

EFFECTSOF DELTAMETHRIN AND BIOALLETHRIN ON THE DENSITIES OF MUSCARINIC RECEPTORS IN THE

CEREBRAL CORTEX AND HIPPOCAMPUS OF NEONATAL MICE’

Control

Deltamethrin Bioallethrin

(m/kg body W (w/kg body ~0

0.7 I 1.2 0.71 12

Cortex

Hippocampus

805 f 60 868 + 78” 815+48 875 f 47” 801 * 66 (17) (15, (13) (15) (13)

9232110 890 -+ 73 854251’ 875 f 77 907 + 65 (18) (15) (13) 04) (11)

“Ten-day-old mice received deltamethrin. bioallethrin, or the vehicle orally once daily for 7 days and were killed 24 hrafter the last administration. [3H]QNB binding(pmol/g protein, mean i SD) was assessed in P2 fractions pooled from two animals. The statistical difference from the control was estimated by Student’s I test.

h p 6 0.02 compared to control. ’ p c 0.05 compared to control. “p < 0.0001 compared to control.

change was observed in the cerebral cortex after the bioallethrin treatment. In the hippocampus no significant change was observed either after deltamethrin or after bioallethrin treatment.

Because the antagonist [3H]QNB cannot distinguish between different muscarinic subpopulations the effect of the pyrethroids on

muscarinic receptors in the cerebral cortex was further investigated in a competition experiment with a muscarinic agonist. The proportions of high-affinity (HA) and low-affinity (LA) binding sites and the affinity constants of the muscarinic receptors were determined in the antagonist ([3H]QNB)/agonist (carbachol) competition assay by using

TABLE 2

EFFECTSOF DELTAMETHRIN AND BIOALLETHRIN ON THE DENSITIESOF NICOTINIC RECEPTORS INTHE

CEREBRAL CORTEX AND HIPPOCAMPUS OF NEONATAL MICE’

Control

Deltamethrin Bioallethtin

(w/kg body W bdkg body wt)

0.71 1.2 0.72 72

cortex 15.9 * 1.5 19.3 + I.Xh 17.5 f 1.9’ 16.1 k2.1 15.7 + 2.1 (15) (15) (13) (15) (13)

Hippocampus 15.5 +- 2.6 17.2 i- 2.3 17.9 -c 1.3 17.9 f 1.3 16.8 i 1.8

(8) (6) (7) (7) (6)

’ Ten-day-old mice received deltamethrin. bioallethrin. or the vehicle orally once daily for 7 days and were killed 34 hr after the last administration. [3H]Nicotine binding (pmol/g protein, mean & SD) was assessed in P2 fractions pooled from two and four animals from the cerebral cortex and hippocampus. respectively. The statistical difference from the control was estimated by Student’s f test.

“1) < 0.0001 compared to control. ’ p < 0.05 compared to control.


TABLE 3

EFFECTS OF DELTAME~HRIN AND BIOALLETHRIN ON HIGH- AND LOW-AFFINITY MUSCARINIC BINDING SITES (76) ANDAFFINITY CONSTANTS(~)INTHECEREBRALCORTEXOFNEONATALMICE'

High-affinity site Low-affinity site

Treatment II %, it- (PM) ‘70 x. (PM)

Control 9 23.1 + 8.4 2.96 76.9 t 8.4 349 Deltamethrin

(0.7 1 mg/kg body wt) 8 33.5 k 10.7” 4.84 66.5 i 10.7h 638” Bioallethrin

(0.72 mg/kg body wt) 8 13.8 f 6Zh 0.77” 86.2 k 6.2” 314

a Ten-day-old mice received deltamethrin, bioallethrin, or the vehicle orally once daily for 7 days and were killed 24 hr after the last administration. The displacement study was performed on P2 fractions pooled from two animals. The binding parameters were estimated from [3H]QNB/carbachol competition curves. The percentage values are means * SD and the affinity constants are geometric means. The statistical evaluation of the percentage values and the affinity constants of the control were made by the Student 1 test and Mann-Whitney Ii test. respectively.

h p < 0.05 compared to control.

different concentrations of carbachol. The two-site model used in a previous experiment (Eriksson and Nordberg, 1986) was used to evaluate the competition data, which are given in Table 3. In mice treated with deltamethrin there was a significant increase and decrease in the percentage of HA and LA binding sites, respectively, and the affinity constant for the LA binding sites was significantly changed. For mice receiving bioallethrin the opposite was seen: i.e., there was a significant decrease and increase in the percentage of HA and LA binding sites, respectively, and the affinity constant for the HA binding sites was significantly changed.

DISCUSSION

Clinical signs of poisoning were only seen in mice receiving the highest doses of deltamethrin (1.2 mg/kg body wt) and bioallethrin (72 mg/kg body wt). The symptoms were typical for the two pyrethroids; i.e., deltamethrin and bioallethrin caused choreoathetosis and whole-body tremor, respectively. The inten- sity and duration of the symptoms declined gradually with each successive day of administration, and they had disappeared com-

pletely by Day 4. This decrease suggests that tolerance had developed, which was not related to a decrease in both the muscarinic and the nicotinic receptors, as reported for adult rats chronically exposed to insecticides such as organophosphorus compounds (see Rus- sell and Overstreet, 1987; Costa, 1988). The differences in the dose used to obtain neurotoxic symptoms between bioallethrin (Type I) and deltamethrin (Type II) in the present study have also been observed in adult rats exposed to deltamethrin and cismethrin (Type I) (Gray and Rickard, 1982). In their study the concentration of deltamethrin in the CNS associated with toxicity was approxi- mately 10% (expressed as amount/brain) of that required for cismethrin. In our study 2% of the dose of bioallethrin (expressed as mg/ kg body wt basis) was required for deltamethrin.

Of the areas of the brain studied, i.e., the cerebral cortex and hippocampus, the lowest doses of deltamethrin and bioallethrin only affected the muscarinic receptors in the cerebral cortex. However, the highest dose of deltamethrin (1.2 mg/kg body wt) also affected the muscarinic receptors in the hippocampus. Further, deltamethrin also affected the


nicotinic receptors in the cerebral cortex. A contributing factor to the difference between Type I and Type II pyrethroids in terms of toxicity and of the symptoms they induce might partly be related to their differential effects on the muscarinic and nicotinic receptors and affected regions of the brain.

The muscarinic receptors in the cerebral cortex were affected by both deltamethrin and bioallethrin. The data from the [3H]- QNB/carbachol binding assay showed that in mice receiving deltamethrin there was an increase in the proportion of HA sites and no significant change in the affinity constant for the HA sites, whereas the affinity constant for the LA sites was significantly changed. The opposite was observed in mice receiving bioallethrin; i.e., the proportion of LA sites increased and the affinity constant for LA sites was not significantly changed, whereas the affinity constant for the HA sites was significantly changed. In immature mice and rats the choline@ system is under rapid development during the neonatal period, and the ontogenesis of the receptors precedes the other variables of the choline@ system (Falkeborn et al., 1983; Marchi et al.. 1983; Hohman and Ebner, 1985). During this development there is an almost parallel increase in the LA and HA sites between the ages of 5 and 20 days (Kuhar et al., 1980). The increased proportion of LA sites and HA sites in bioallethrin- and deltamethrin-treated mice, respectively, might have been due to synthesis of these subpopulations of muscarinic receptors.

The bioallethrin exposure (0.72 mg/kg body wt) resulted in increases in the specific [3H]QNB binding sites and in the proportion of low-affinity binding sites in the cerebral cortex. This increase in both [3H]QNB binding sites and LA sites in the immature mouse cerebral cortex is in accordance with the effect seen earlier after a single low dose of DDT (0.5 mg (I .4 pmol)/kg body wt) was given to lo-day-old mice (Eriksson and Nordberg, 1986). Similarities in effects between pyrethroids (Type I) and DDT have

also been observed in nerve membrane preparations; i.e., both prolong the open time of the sodium channels on the axon which leads to an increased neuronal activity (Van den Bercken and Vijverberg, 1980; Vijverberg et al., 1982; Narahashi, 1982; Lund and Nara- hashi, 1983). Changes in receptors are usually reciprocal to changes in stimulation, result- ing in a pattern consistent with compensatory adaptation to the change in stimulation (Creese and Sibley, 198 1). Exposure to bioallethrin for 1 week may generate repetitive fir- ing, leading to a reduction in the content of the transmitter ACh. The observed increase in the density of muscarinic receptors might therefore be a compensatory mechanism to cope with an altered ACh turnover.

In conclusion, this study shows that two pyrethroids of different types, deltamethrin, an a-cyanophenoxybenzyl containing pyrethroid, and bioallethrin, a noncyano containing pyrethroid, affect the cholinergic system in the neonatal mouse brain in two different ways. At doses below those causing neurotoxic symptoms both affect the muscarinic receptors in the cerebral cortex, with deltamethrin causing an increase and a decrease in the HA and LA binding sites, respectively, and with the reverse occurring after bioallethrin treatment. Deltamethrin also affected the nicotinic receptors in the cerebral cortex, which were manifested as an increase in the specific [3H]nicotine binding sites. This study further supports that the cholinergic system under rapid development in the neonatal mouse brain is sensitive to environmental pollutants as earlier reported for DDT and DDOH-palmitic acid (Eriksson and Nord- berg, 1986) and for 3,4,3’,4’-tetrachlorobiphe- nyl (Eriksson, 1988).

ACKNOWLEDGMENTS

The authors thank Dr. Tessier. Rouse11 Uclaf. France. for the gift of deltamethrin and bioallethrin and Miss Anna Pettersson for excellent technical assistance. This work was financially supported by grants from the Swed-


ish Environmental Protection Board and the Bank of Sweden Tercentenary Foundation.

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Effects of two pyrethroids, bioallethrin and deltamethrin, on subpopulations of muscarinic and nicotinic receptors in the neonatal mouse brain