TOXICOLOGY AND APPLIED PHARMACOLOGY 85, 12 1 - 127 ( 1986)

The Effects of DDT, DDOH-Palmitic Acid, and a Chlorinated Paraffin on Muscarinic Receptors and the Sodium-Dependent Choline Uptake

in the Central Nervous System of Immature Mice

PERERIKSSONANDAGNETANORDBERG*

Department of Zoophysiology, Uppsala University, Box560, S-751 22 Uppsala. Sweden, and *Department of Pharmacology, Uppsala University, Box 5 73.9 75123 Uppsala, Sweden

Received January 3.1986: accepted June 2, I986

The Effects of DDT, DDOH-Palmitic Acid, and a Chlorinated Paraffin on Muscatinic Recep- tors and the Sodium-Dependent Choline Uptake in the Central Nervous System of Immature Mice. ERIKSSON, P., AND NORDBERG, A. ( 1986). Toxicol. Appl. Pharmacol. 85, 12 1 - 127. Ten- day-old NMRI mice were given a single peroral dose of 1.4 pmol/kg body wt of either of the substances of DDT (0.5 mg), DDOH-PA (a DDT metabolite conjugated to palmitic acid, 0.7 mg). or polychlorohexadecane (PCHD, a synthesized chlorinated paraffin; I .O mg). The mice were killed either 24 hr or 7 days after treatment, and crude synaptosomal fractions (P2) were prepared from the cerebral cortex and hippocampus. The density of the muscarinic receptors was assayed by measuring quinuclidinyl benzilate ([3H]QNB) specifically bound in the P2 frac- tion. A significant increase in the specific t3H]QNB binding was observed in the cerebral cortex in DDT- and DDOH-PA-treated mice 7 days after treatment, compared to control. These re- sults were further explored by determining the ratio of high- and low-affinity binding sites by using an agonist (carbachol)-antagonist ([3H]QNB) competition assay. A significant decrease in the percentage of high-affinity binding sites and a corresponding increase in the percentage of low-affinity binding sites, compared to control, were observed after DDT and DDOH-PA treat- ment. The presynaptically sodium-dependent choline uptake system also was studied. In mice receiving PCHD there was a significant decrease (65%) in the V,,,, value 7 days after treatment, but no change was observed in mice receiving DDT or DDOH-PA. This study shows that the sensitivity of the choline@ system to persistent xenobiotics acting over a long period of time may be higher in the immature mouse. 0 1986 Academic PI=, Inc.

INTRODUCTION

Many of the chlorinated hydrocarbons are present in the environment as persistent con- taminants. Recent studies have shown that some of these substances, 1 , 1,l -trichloro-2,2- his@-chlorophenyl)ethane (DDT) and a chlorinated paraffin (CP), polychlorohexa- decane (PCHD), are retained to a greater ex- tent in the mouse brain when given to pre- weanling mice at the age of 10 days (Eriksson, 1984; Eriksson and Damerud, 1985). In mice this point of time coincides with rapid devel- opment and maturation of the brain, includ-

ing axonal and dendritic growth, establish- ment of neural connections, and acquisition of new motor and sensory qualities (Bolles and Woods, 1964; Davison and Dobbing, 1968).

The retained substances may be in either unmetabolized or metabolized form. Leighty et al. (1980) have found a DDT metabolite, DDOH, conjugated to fatty acids in the liver of DDT-treated female rats. They have pro- posed that such conjugates may be one possi- ble form for metabolized substances to be retained in the body. These highly lipophilic conjugates might be transferred from the

121 0041-008x/86 $3.00 Copyright 0 1986 by Academic Press. Inc. All rights of reproduction m any form reserved.

122 ERIKSSON AND NORDBERG

mother to the offspring via the milk. as it is known to occur with many other lipo- philic chlorinated hydrocarbons, e.g., DDT, CP, and polychlorinated biphenyls (PCBs) (Woolley and Talens, 197 1; Takagi et al., 1976; Vodicnik and Lech, 1980; Darnerud, personal communication).

DDT is a well-known insecticide with neu- rotoxic effects (reviewed by Hayes, 1959; Herr et ai.. 1985; Tilson et al., 1985) and CPs are industrial contaminants which have been shown to give neurotoxic symptoms in mam- mals (Eriksson and Kilhstrom, 1985). Some of the possible neurochemical mechanisms underlying the neurotoxic effects of DDT in- clude alterations in the levels of different brain transmitters or their metabolites, e.g., acetylcholine (ACh), norepinephrine, seroto- nin, and GABA (reviewed by Hrdina et al., 1975: Woolley, 1982). One primary effect of DDT seems to be its interaction with the so- dium channels, leading to increased neuronal activity (Narahashi, 1982).

During the maturation of the CNS there are age-related changes in the concentration of specific cholinergic receptors in rodents (Coyle and Yamamura, 1976; Falkebom et aI., 1983), and in a previous study we have found an effect of a single low dose of DDT on muscarinic receptors, but not on nicotinic receptors, in the brain of the immature mouse (Eriksson et al., 1984).

The present study was undertaken to ex- amine further the effects of a low dose of DDT, and also of PCHD and DDOH conju- gated to palmitic acid (DDOH-PA), on the muscarinic receptors in the brain of the im- mature mouse. The sodium-dependent cho- line uptake system also was measured, since the uptake can be used as a marker for the synthesis and turnover of ACh in the brain (Nordberg, 1978).

METHODS

Ten-day-old NMRI mice of both sexes. which were offspring either of pregnant mice purchased direct from ALAB. Sweden (used in the muscarinic receptor assay)

or of mice from our own department mated with animals from the same breeder (used in the choline uptake study). 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 (Ewos. Sweden) and tap water ad libitum.

DDT (puriss. Fluka AC. Switzerland). DDOH-pal- mitic acid (a gift from Professor K. Olsson. Department ofchemistry and Molecular Biology. Swedish University of Agricultural Sciences, Sweden), and PCHD (a gift from Dr. A. Bergman. Section of Organic Chemistry. Wallet&erg Laboratory, University of Stockholm. Swe- den) were separately dissolved in a mixture ofegg lecithin (Merck) and peanut oil (Oleum arachidis) (1: 10 w:w). The solutions were then sonicated together with water to yield a 20% (w:w) fat emulsion vehicle containing 0.05 mg DDT, 0.07 mg DDOH-PA. and 1 .O mg PCHD/ml. respectively, according to a method described by Bruns- tram and &berg (I 982). except for the use of dichloro- methane instead of diethylether and excluding auto- claving.

The mice received a single administration of 1.4 pmol/ kg body wt per OS of one ofthe substances, corresponding to 0.5 mg DDT, 0.7 mg DDOH-PA. and 1 .O mg PCHD/ kg body wt, respectively. via a PVC tube (diameter 1 .O mm). Mice serving as controls received in the same man- ner 10 ml/kg body wt of the 20% fat emulsion vehicle.

The animals were killed by decapitation 24 hr or 7 days after treatment. The brains were rapidly removed and dissected on an ice-cold glass plate into cerebra1 cortex and hippocampus(Glowinski and Iversen, 1966) and im- mediately placed in ice-cold sucrose (0.32 M). The brain tissues from mice killed 24 hr and 7 days after treatment were pooled from three and two animals, respectively, and homogenized in 20 and 25 vol. respectively. of ice- cold sucrose with a Potter-Elvehjem homogenizer ( IO up-and-down strokes. 900 rpm). The homogenate was centrifuged at IOOOgfor 10 min and the supematant fur- ther centrifuged at 17,OOOg for 15 min. The resulting pel- let was suspended and homogenized in the original vol- ume ofthe ice-cold sucrose to yield a crude synaptosomal fraction, P2 (Gray and Whittaker. 1962), with a protein content of about 2 mg/ml. determined by the Lowry method (Lowry et a/., 195 1) using bovine serum albumin as standard. The P2 fractions were kept frozen (~30°C) until the muscarinic receptor density assay was per- formed (within 2 months), and thereafter stored at -7o’C until the measurement of high- and low-affinity muscarinic binding sites was performed (within 6 months). The P2 fractions to be used in the high-affinity choline uptake study were kept on ice until the onset of incubation (within 30 min).

Measurement of the muscarinic receptor density was performed by using the tritium-labeled muscat-uric an- tagonist, quinuclidinyl benzilate ([‘HIQNB: sp act 1.59

NEUROTOXICITY OF IMMATURE MICE 123

TBq/mmol, Amersham International plc, U.K.) accord- ing to the method of Nordberg and Winblad (1981). An aliquot of the P2 fraction (75 ~1) was incubated with [3H]QNB (20 ~1, 0.2 nM) for 60 min at 25°C in 50 mM NaKP04 buffer (pH 7.4) in a total volume of 1.02 ml. In parallel samples atropine (20 ~1, 10m4 M) was present for measuring the nonspecific binding. Each binding was de- termined in duplicate. The specific binding was deter- mined as the difference ofthe amount bound in the pres- ence and in the absence of atropine. The specific binding constitutes about 98% of the total [3H]QNB binding.

Measurement of the muscarinic high- and low-affinity binding sites was performed by using the muscarinic ago- nist carbachol (carbamylcholine chloride, Sigma) follow- ing the method of Nordberg and Wahlstrom (1982). Different concentrations of nonlabeled carbachol(20 ~1, IO~‘-lO~~* M) were present in the incubation medium. The concentration of [‘HIQNB was changed to 0. I nM. Each binding was determined in triplicate. The data of the competitive displacement of [3H]QNB by carbachol were fitted by a nonlinear least-squares method as de- scribed by Birdsall et al. (I 978) and by Bartfai and Hed- lund ( 1979). The following model by Bartfai and Hed- hind was used:

where ?’ stands for the amount of [‘H]QNB bound at different concentrations of carbachol (s), and I’, and V, stand for the percentage of high- and low-affinity binding sites with the corresponding affinity constants /(I and k2.

Measurement of the sodium-dependent choline up- take system was performed by using [3H]choline (sp act 2.89 TBq/mmol, Amersham International plc. U.K.) by the method described by Simon et al. (1976) and Nord- berg (1978). Aliquots of the P2 fractions (100 ~1) were preincubated in both a Krebs-Ringer phosphate me- dium (900 ~1; NaCl 126 mM, Na2HP04 15.8 mM, KCI 4.75 mM, CaC& 1.27 mM. MgClz 1.42 mM. dextrose 2 mg/ml) at pH 7.4 and a sodium-free medium where tris- phosphate 15.8 mM and sucrose 252 mM replaced Na2HPOJ and NaCl, for 5 min at 37°C. After addition of nonlabeled and labeled choline (20 ,~l. 18.5 kBq), giving a final concentration of 0.05-0.5 pM choline in the me- dium, the incubation was terminated after 4 min. Each sample was determined in duplicate. The sodium-depen- dent uptake ofcholine was determined by subtracting the amount obtained with the sodium-free medium from the uptake obtained with the medium containing sodium. The kinetic parameters Km and r,, were obtained by Eadie-Hofstee plots.

All incubations were carried out in plastic microfuge tubes (I .5 ml, Eppendort). The incubations were termi- nated by placing the tubes in an ice-cold bath followed by centrifugation at 22,000g for 5 min. Each supematant was discharged and the pellet surface was washed with ice-cold buffer (sodium-free in the choline uptake study)

1 ‘ON

f

- 1 TROL DOT ODOH-PA PCHD CONTF

FIG. 1. [3H]QNB binding (pmol/g protein) in cerebral cortex 24 hr and 7 days after a single administration of either DDT (0.5 mg). DDOH-PA (0.7 mg), PCHD (1.0 mg), or the vehicle (IO ml) per kg body wt. per OS, to lo- day-old mice. The brain tissues from 1 I- and I7-day-old mice were pooled from three and two animals. respec- tively. The height of the bars represents means t SD of the specific binding of [3H]QNB which was carried out according to Nordberg and Winblad (198 1). The statisti- cal difference from the control was estimated by Stu- dent’s t test.

and drained. The tips of the tubes containing the pellets were then cut into mini scintillation vials and dissolved overnight in 1 ml Soluene 350 (Packard)/toluene (1:3). After 4 ml Insta-fluor scintillation liquid (Packard) was added, the samples were precooled to 4°C and placed in the dark overnight before counting in liquid scintillation spectrometer (LKB Wallac 81000). The counting effi- ciency was about 35% and the quench was corrected by using the external standard method.

The statistical difference between controls and treated animals in the receptor density study was assessed with Student’s t test. The difference in the affinity constants and the proportions of binding sites between controls and treated animals was evaluated by using Mann-Whit- ney U test and Student’s t test, respectively.

RESULTS

In animals receiving DDT and DDOH- PA, there was a significant increase in the amount of specific [3H]QNB binding sites in the cerebral cortex 7 days after treatment, compared to control animals (Fig. 1). No sig- nificant change was observed 24 hr after treatment. In the hippocampus no change was noticed either 24 hr or 7 days after treat- ment. Administration of PCHD to the lo- day-old mice caused no significant alteration

124 ERIKSSON AND NORDBERG

TABLE 1

THE EFFECT OF DDT, DDOH-PA, AND PCHD ON HIGH- AND LOW-AFFINITY MUSCARINIC BINDING SITES (96) AND AF?=INITY CONSTANTS (k) IN THE CEREBRAL CORTEX OF IMMATURE MICE’

High-affinity site Low-affinity site

Treatment n % k (PM) % k (PM)

Control 10 15.422.9 0.57 80.6 2 3.1 276

DDT 9 11.4~12.1~ 1.22 84.4 f 2.4' 298

DDOH-PA 10 12.3? 3.5d 0.67 83.3 k 3.4' 270 PCHD 8 15.622.5 0.73 80.9 k 2.0 259

‘Ten-day-old mice received a single dose of either DDT (0.5 mg), DDOH-PA (0.7 mg), PCHD (1 .O mg), or the vehicle (IO ml) per kg body wt per OS, and were killed 7 days later. The binding parameters were estimated from [“H]QNB/carbachol competi- tion curves (see Methods for details). The % values are means ? SD and the affinity constants are geometric means. The statisti- cal evaluation of the % values and the affinity constants to the control were made by Student’s t test and Mann-Whitney U test, respectively.

bp < 0.005 compared to control. ‘p < 0.01 compared to control. dp < 0.05 compared to control. ’ 0.05 < p < 0.1 compared to control.

in the specific [3H]QNB binding sites in the two brain fractions studied.

The observed changes in the specific [3H]QNB binding in the cerebral cortex was further investigated. In the antagonist ([3H]QNB)/agonist (carbachol) competition assay, the proportion of high- and low-affinity binding sites and affinity constants of the muscarinic receptors was evaluated by using different concentrations of carbachol. Con- sidering the experimental design (the choice of carbachol concentrations) and the results obtained from the goodness of fit of the total material for each respective treatment, the two-site model was the most suitable for eval- uating the competition data. As seen in Table 1, there was a significant decrease in the per- centage of high-affinity binding sites in mice receiving DDT and DDOH-PA, a significant increase in low affinity in those receiving DDT, and a tendency toward an increase in those receiving DDOH-PA, compared to the control mice. No significant changes in the affinity constants could be noted. No changes

were observed between the control and the PCHD-treated mice.

The evaluation of the goodness of fit of the total material for each respective treatment to one-, two-, and three-site models was based on the “extra sum of squares” principle (Draper and Smith, 1966, as referred to by Munson and Rodbard, 1980). When using the three-site model, a significant increase was seen in animals constituting the control group and the PCHD group, but not in the DDT or DDOH-PA groups (Table 2). The existence of super-high affinity-binding sites in the control and PCHD-treated mice but not in the DDT- and DDOH-PA-treated ani- mals shows that DDT and DDOH-PA treat- ment shifts the binding sites toward the low- affinity part.

Table 3 shows the effect of DDT, DDOH- PA, and PCHD on the sodium-dependent choline uptake system in the P2 fractions. A significant change, a 65% decrease in I’,,,, is observed in mice receiving PCHD, compared to control. Neither DDT nor DDOH-PA caused any changes in the kinetic parameters v,,, or G.

DISCUSSION

This study shows that DDT and its conju- gated metabolite, DDOH-PA, at a low dose (0.5 and 0.7 mg/kg body wt, respectively),

TABLE 2

THE GOODNESS OF FIT OF THE [‘H]QNB/CARBA- CHOL BINDING DATA OF THE TOTAL MATERIAL OF EACH RESPECTIVE TREATMENTS

Compared site model Control DDT DDOH-PA PCHD

1 versus 2 p < 0.01 p<o.o1 p < 0.01 p<O.Ol 2versus3 pio.01 p> 0.05 p > 0.05 p<O.Ol

y The total material of each respective treatment was fitted by using the nonlinear least-squares method, and the goodness of fit is based on the “extra sum of squares” principle (Draper and Smith, 1966, as referred to by Munson and Rodbard, 1980). The p values are obtained from the tabulated Fdistribution.

NEUROTOXICITY OF IMMATURE MICE 125

TABLE 3

THEEFFECTOFDDT,DDOH-PA, AND PCHD ONTHESODIUM-DEPENDENTCHOLINE UPTAKE INTHECEREBRALCORTEXOFIMMATURE MICE’

Control (n = 9)

DDT DDOH-PA (n = 10) (n = 10)

PCHD (n = 6)

c-m,, 26.5 ?I.8 29.4 f 11.0 28.9 f7.4 17.2 z!z 5.0h

Km 0.46-t-0.11 0.52 + 0.21 0.51 kO.16 0.44kO.16

ci Ten-day-old mice received a single dose of either DDT (OS mg), DDOH-PA (0.7 mg), PCHD (1 .O mg). or the vehicle (10 ml) per kg body wt per OS, and were killed 7 days later. The P2 fractions were incubated in different choline concentrations (0.05-0.5 FM) and [3H]choline (18.5 kBq) for 4 min. The kinetic parameters, V,, (pmol. mg protein -‘. 4 min-I) and Km (fiM). were obtained by Eadie-Hofstee plot and represent means f SD. p values were calculated by Student’s f test.

” p < 0.05 compared to control.

which for DDT is of physiological interest (Sl- orach and Vaz, 1983), affect the muscarinic cholinergic receptors in the brain of the im- mature mouse. The dose used also causes re- gional differences within the brain since the cerebral cortex was affected but not the hip- pocampus. The increased binding of the an- tagonist [3H]QNB, observed in the cerebral cortex fraction 7 days after treatment, is in agreement with previous results (Eriksson et al., 1984). The change is further supported in the antagonist/agonist competition experi- ment, where a subdivision into sites with different affinities is obtained. Here the pro- portion of low-affinity binding sites (corre- sponding to type M,) was significantly in- creased and the high affinity (corresponding to type M2) decreased in the cerebral cortex 7 days after treatment. This change can be of physiological importance since the low- affinity part seems to be associated with the physiological response (McKinney and Rich- elson, 1984).

Both the increased antagonist binding and the increased part of low-affinity sites, which are seen after DDT exposure, might be due to changes within the neuronal membrane. It is known from studies on isolated nerve fiber preparations that DDT exposure prolongs the open time of sodium channels on the ax- ons, which leads to an increased neuronal ac- tivity (Narahashi, 1982). Concerning the du-

ration of exposure, previous investigations have shown that lo-day-old mice receiving [14C]DDT held as much radioactivity 7 days after treatment as after 24 hr after treatment (Eriksson, 1984), and the radioactivity was also found to be evenly distributed within the brain (Eriksson and Darnerud, 1985). This implies that the neurons will be constantly in- fluenced for 1 week and that repetitive dis- charges may be generated, causing a reduc- tion in neurotransmitter content. A decrease in the level of ACh in the cerebral cortex also has been observed in adult rats exposed to 5 mg DDT/kg body wt im for 45 days (Hrdina et al., 1972). Furthermore, in the present study no change was observed in the sodium- dependent choline uptake system in mice re- ceiving DDT. There are reports that support a link between the sodium-dependent cho- line uptake system and the neural impulse flow (Kuhar et a!., 1973; Simon et ul., 1976) and a linear relationship between this uptake and the synthesis of ACh has been shown in the mouse brain (Nordberg, 1978). The lack of effect on the sodium-dependent choline uptake system may be due to the fact that in the 17-day-old mouse, this system has only reached about 50- 60% of the adult level (ex- trapolation from rats, Coyle and Yamamura, 1976). and an increase in capacity to cope with an increased impulse flow may not be possible. The muscarinic receptors have,

126 ERIKSSON AND NORDBERG

however, reached the adult level in the 17- day-old mouse (Falkeborn et al., 1983). The observed increase in antagonist binding and increase in low-affinity binding sites can therefore be a compensatory mechanism to cope with an altered ACh turnover, which is known to occur in adult rodents exposed to xenobiotics (Burgen, 1982; Nordberg and WahlstrGm, 1982).

Another interpretation is that the increase in the low-affinity binding sites might in- crease the sensitivity of the muscarinic sys- tem leading to increased motor activity, known in supersensitivized rodents (e.g., Ma- jocha and Baldessarini, 1984), or even cause convulsions (Nordberg and Wahlstrtim, 1982).

An interesting finding was the effect of DDOH-PA on the muscarinic receptors. The origin of this effect may differ from that of DDT, but DDOH-PA seems nearly as potent as DDT in altering the muscarinic receptors. That a fatty acid conjugate of a DDT metabo- lite, orally given to preweanling mice, has an effect similar to that of the parent compound has to our knowledge not previously been shown. The proportion of fatty acid-conju- gated metabolites in relation to the parent compound is not known but they are known to be retained in vivo, which also is demon- strated for cannabinoids (Leighty et al., 1976; Leighty et al., 1980).

In contrast to the other two substances, PCHD had no effect on the amount of mus- carinic receptors while a decrease in the sodi- um-dependent uptake of choline, by 65% in ~‘nlax, was measured. This finding indicates that PCHD primarily has a presynaptic effect.

The present findings-that a single dose of DDT, DDOH-PA, or PCHD affects the cho- linergic system in the immature mouse brain-imply that the sensitivity to persistent xenobiotics acting over a long period of time may be higher in the preweanling mouse. Furthermore, fatty acid conjugates of metab- olized environmental pollutants, having a potency similar to the parent compound and

which may be transferred to offspring during the preweaning period, require further study.

ACKNOWLEDGMENTS

The authors are indebted to Dr. Ake Bergman and Professor Kjell Olsson for the synthesis of polychloro- hexadecane and DDOH-palmitic acid, respectively. This work was financially supported by grants from the Swedish Council for Planning and Coordination of Re- search to Professor Jan Erik KihlstrGm and from Stif- telsen Lars Hiertas Minne.

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