Toxicology, 77 (1993) 21-30 21 Elsevier Scientific Publishers Ireland Ltd.

Neonatal exposure to DDT induces increased susceptibility to pyrethroid (bioallethrin) exposure at

adult a g e . - Changes in cholinergic muscarinic receptor and behavioural variables

Per Eriksson a, Ulrika Johansson a, Jonas Ahlbom a and Anders Fredriksson b

aDepartment of Zoophysiology, Uppsala University, Box 560, S-751 22 Uppsala and bDepartment of Toxicology, Uppsala University, Box 594, S-751 24 Uppsala (Sweden)

(Received February 10th, 1992; accepted September 12th, 1992)

Summary

We have recently reported that DDT and the pyrethroid bioallethrin cause similar changes in the brain muscarinic cholinergic receptors (MAChR) and behavioural disturbances in the neonatal and adult mouse when given to neonatal mice during the peak of rapid brain growth. In the present study the in- teraction between neonatal and adult exposure to DDT and bioallethrin, respectively, is explored. Ten- day-old NMRI mice received a single low oral dose of DDT (0.5 mg/kg body wt). At adult age (5 months) the mice received bioallethrin 0.7 mg/kg body wt./day per os for 7 days. Mice used as controls received a 20% fat emulsion vehicle. The spontaneous behavioural tests revealed significant differences, both in mice treated neonatally with DDT and receiving bioaUethrin as adults and in mice receiving the vehicle as neonates and bioallethrin as adults, compared with their corresponding controls. However, the behavioural changes developed in mutually opposite directions. Significant changes in MAChR, assayed in the P2 fraction of the cerebral cortex by using the muscarinic antagonist, quinuclidinyl benzilate ([3H]QNB) and agonist carbachol, was only observed in animals receiving DDT as neonates and bioallethrin as adults. The present study indicates an increased susceptibility in the cholinerglc muscarinic receptors and a different behaviour reaction in animals already exposed to DDT (at a physiologically relevant dose), when again exposed to a similar neurotoxic agent as adults.

Key words: Neurotoxicity; 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)-ethane (DDT); Pyrethroid; Interac- tion; Behaviour; Muscarinic receptors

Introduction

Many of the environmental pollutants have become known as persistent environmental contaminants. One of these chemicals is DDT (1,1,1-trichloro-2,2- bis(p-chlorophenyl)-ethane), a chlorinated hydrocarbon, which is one of the best

Correspondence to: Per Eriksson, Department of Zoophysiology, Uppsala University, Box 560, S-75122 Uppsala, Sweden.

0300-483X/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

22

known insecticides. Because of its solubility in fat and resistance to breakdown under environmental conditions, it accumulates in food chains, is stored in the living organism [1,2] and can be transferred from mother to offspring via the milk [3,4].

In recent years the pyrethroids, derivatives of natural pyrethrins [5], have emerged as a very useful class of insecticides. By virtue of their high insect/mammal toxicity ratio, rapid detoxication in mammals and absence of cumulative toxicity [6,7] they have replaced many of the earlier compounds and have therefore achieved a widespread agricultural and environmental health application. The pyrethroids are commonly divided into Type I compounds, which lack an et-cyano substituent and Type II compounds, which contain an o~-cyanophenoxybenzyl substituent [8,9]. The mechanism of action of DDT and the pyrethroids (Type I) appears to be a specific effect on nerve membrane sodium channels whereby both prolong the membrane sodium current time and thereby increase the depolarizing after-potential which causes repetitive discharges [10-14].

During perinatal brain growth - - 'the brain growth spurt' [15], the cholinergic transmitter system undergoes rapid development in the neonatal rodent [16-20]. The cholinergic system is connected with many behavioural phenomena [21] and the muscarinic cholinergic receptors (MAChR) in the cerebral cortex are suggested to be involved with higher cognitive functions [22,23]. We have recently found the developing cholinergic system to be sensitive to environmental toxicants such as DDT, pyrethroids, Polychlorinated biphenyls (PCB) and nicotine [24,25,27-30]. These experiments have shown that DDT and the pyrethroid, bioallethrin, cause the same changes in MAChR variables in the cerebral cortex and in behaviour in both neonatal and adult mice following neonatal exposure to these substances [24,25,27,28]. Further, these aberrations in the adult mouse after neonatal exposure to DDT are not correlated to the presence of DDT in the adult brain [26].

The combined effects between early (during lactation) and late (adult) exposure to persistent and non-persitent agents, which is likely to occur in nature, has not re- ceived much attention. Since bioallethrin (Type I, pyrethroid) and DDT show the described similarities with respect to both neuroreceptor and behavioural changes, the present study was undertaken to ascertain whether neonatal exposure to a low dose of DDT could potentiate and/or modify the reaction to an adult exposure with a short-acting insecticide. Would the adult mice neonatally exposed to DDT show a greater susceptibility to a new exposure than the control mice?

Methods

Pregnant NMRI mice were purchased from ALAB, Sollentuna, Sweden. Each lit- ter, adjusted within 48 h to 8-10 mice and to contain offspring of either sex in about equal number, was kept together with its respective mother in a plastic cage in a room at a temperature of 22°C and a 12/12-h light/dark cycle. At the age of 4 weeks the mice were weaned and the males were placed and raised in groups of 5-8 in a room for male mice only. The animals were supplied with standardized pellet food and tap water ad libitum. DDT (Puriss, Fluka AG, Switzerland) and bioallethrin (2-allyl-4-hydroxy-3-methyl-2-cyclopenten-l-one-(1R,3R, 4S)-2,2- dimethylcyclopro- pane carboxylate, Roussel Uclaf, France) were dissolved separately in a mixture of

23

egg lecithin (Merck, Darmstadt, Germany) and peanut oil (Oleum arachidis) (1:10 w/w) and were thereafter sonicated together with water to yield a 20% (w/w) fat emulsion vehicle containing DDT at a concentration of 0.05 mg/ml, or bioallethrin at a concentration of 0.180 mg/ml. Ten-day-old mice received a single dose of 0.5 mg (1.4 #mol) DDT/kg body wt. per os, via a PVC tube (diameter 1.0 mm). Mice serving as controls received in the same manner 10 ml/kg body wt. of the 20% fat emulsion vehicle. At the age of 5 months, bioallethrin was administered orally via a metallic gastric tube, as one single dose per day for 7 days. The amount of bioallethrin given was 0.7 mg (2.4 #mol)/kg body wt. Mice serving as controls received in the same manner 10 ml/kg body wt. of the 20% fat emulsion vehicle. Each treatment group consisted of mice from three to four different litters [31].

Behavioural testing Spontaneous behaviour was tested in male mice 24 h after the last dose of

bioallethrin. The animals were only tested once and the tests were performed be- tween 08:00-12:00 h under the same light and temperature condition as the housing. Nine mice, taken from three to four different litters, were tested from each treatment group. Motor activity was measured for 3 x 20 min in an automated device con- sisting of cages (40 x 25 x 15 cm) placed within two series of infrared beams (low level and high level) (Rat-O-Matic, ADEA Elektronik AB, Uppsala, Sweden) [32].

Locomotion. Counting occurred when the mouse moved horizontally through the low-level grid of infrared beams.

Rearing. Vertical movement was registered at a rate of 4 counts per s, as long as a single high level beam was interrupted, i.e. the number of counts obtained was pro- portional to time spent rearing.

Total activity. Registered by a pick-up (mounted on a lever with a counterweight) with which the test cage was in contact. The pick-up registered all types of vibration within the test cage, i.e. those caused by mouse movements, shaking (tremors) and grooming.

Receptor assay The mice were killed by decapitation 48 h after the behavioural tests. Brains were

dissected onto an ice-cold glass plate and the cerebral cortex was immediately placed in 30 times its own weight of ice-cold sucrose (0.32 M) and homogenized with a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 1000 x g for 10 rain and the supernatant further centrifuged at 17 000 x g for 15 min. The resulting pellet was suspended and homogenized in the original volume of ice-cold sucrose to yield a crude synaptosomal fraction, P2 [33], with a protein content of about 2 mg/ml, determined by the Lowry method [34]. The P2 fractions were kept frozen (-25°C) until assayed (within 4 months).

Measurement of the muscarinic receptor density was performed by using the tritium-labeled muscarinic antagonist quinuclidinylbenzilate ([3H]QNB, spec. act. 1.54 TBq/mmol, Amersham International, Amersham, Bucks., UK) as described elsewhere [24,35]. The P2 fraction (75 #1) was incubated with [3HIQNB (20/A, 0.2 riM) for 90 min at 25°C in 50 mM NaKPO4 buffer (pH 7.4) in a total volume of 1.02 ml. In parallel samples, atropine (20 #l, l0 -4 M) was present for measuring the

24

non-specific binding. Specific binding was determined as the difference in the amount bound in the presence and in the absence of atropine. Specific binding con- stitutes about 98% of the total [3H]QNB binding. Measurement of the muscarinic high-affinity (HA) and low-affinity (LA) binding sites was performed in a displace- ment study by using different concentrations of carbachol (10 -2 to 10 -s M) [36,37] and [3HIQNB, 0.1 nM. The data of the competitive displacement of [3HIQNB by carbachol were fitted by a non-linear least-squares method as described by Birdsall et al. [36]. The computerized model in Graph Pad was used to calculate the percen- tages of HA- and LA-binding sites with corresponding affinity constants.

Statistical analysis Behavioural data were analysed using ANOVA with a split-plot design [38]. Pair-

wise testing between treated groups and their corresponding control groups was per- formed with the Tukey HSD tests [38].

Muscarinic receptors. Student's t-test was used to evaluate differences between controls and treated animals, except for the evaluation of the affinity constants, where the Mann-Whitney U-test was used.

R ~

The results concerning 'locomotion', 'rearing' and 'total activity' variables in 5- month-old NMRI male mice after a single low dose of 0.5 mg/kg body wt. per os of DDT at the age of 10 days and a dose of 0.7 mg/kg body wt. per os for 7 days at the age of 5 months, or controls receiving the 20% fat emulsion vehicle at a cor- responding age are given in Fig. 1. Split-plot ANOVA indicated significant group × period interactions [F(6,64)] = 24.2; F(6,64) = 27.68; F(6,64) = 22.54] for 'locomotion', 'rearing' and 'total activity', respectively. The test revealed significant differences both in mice treated on day 10 with DDT and receiving bioallethrin as adults and in mice receiving vehicle at the age of 10 days and bioallethrin as adults, compared with their respective controls. The two sets of results were totally inverted, however. During the first 20-rain period, animals exposed to DDT on day 10 and receiving bioallethrin as adults showed a hyperactive condition in the variables 'locomotion' and 'total activity' and a hypoactive condition in the variable 'rearing', compared with their controls, whereas the animals exposed to the vehicle on day 10 and receiving bioallethrin as adults showed a hypoactive condition in 'locomotion' and 'total activity' and a hyperactive condition in 'rearing', compared with their con- trols. Further, mice exposed to DDT on day 10 and bioallethrin as adults displayed a rearing pattern opposite to all other treatments. The quota 40-60 rain over 0-20 rain is over 1, compared to below 1 in the other groups.

Mice exposed to the vehicle on day 10 and receiving bioallethrin as adults showed a hypoactive condition for the 'locomotion' variable during the first 20-min period and a hyperactive condition during the last 20-rain period (40-60 rain).

Mice exposed to DDT on day 10 and receiving the vehicle as adults displayed dur- ing the last two 20-rain periods (20-40 min and 40-60 rain) a hyperactive condition in all three test variables.

The animals were killed 48 h after the behavioural tests and the MAChR in the

700-

~ 600.

500.

400.

°0200. Oj 100,

c o

1 2 3 4 1 2 3 4 1 2 3 4

50O

,oo 0

8500

_• 3000

25OO

20oo

,ooo

5O0

I

C

x x ~ x B

n 2 : 3 4 1

II

1.ii 1 2 3 4 1

0 - 2 0

o

\ \ \ \ ~ x x x '

~ x x . x ~ x \ x "

2 3 4

C

2 3 4

2 0 - 4 0

I

1 2

I

c

8 4

c

2 3 4

4 0 - 8 0

I

25

TIME (rain)

Fig. 1. Spontaneous behaviour in 5-month-old NMRI male mice after they had received at the age of 10 days a single dose of DDT (0.5 rag) or the vehicle (10 ml, per kg body wt. per os) and at the age of 5 months, bioallethrin (0.7 mg) or the vehicle (10 ml per kg body wt. per os) once daily for 7 days. For measurement of motor activity, see Methods. Statistical analysis of behavioural data was performed using ANOVA with a split-plot design [38]. There were significant group × period interactions [F(6,64) = 24.2; F(6,64) = 27.68; F(6,64) = 22.54] for the variables 'locomotion', 'rearing' and 'total activity', respectively. Pairwise testing between treated groups and control groups was performed with the Tukey HSD tests (alpha = 0.01) [38]. The treatment groups are indicated by: 1, control-control; 2, controi-bioallethrin; 3, DDT-control; 4, DDT-bioallethrin. A, Significant difference between 1 and 2; B, significant difference between 3 and 4; C, significant difference between 1 and 3.

26

cerebral cortex were assayed. The densities of the MAChR are given in Table I. In animals exposed to DDT on day 10 and receiving bioallethrin as adults, there was a significant increase in the amount of specific [3H]QNB binding sites, compared with control animals (mice neonatally exposed to DDT receiving vehicle as adults). No significant changes were observed in mice exposed to the vehicle on day 10 and receiving bioallethrin as adults, compared with their control animals.

The observed changes in the specific [3H]QNB in the cerebral cortex were further investigated. In the antagonist ([3H]QNB)/agonist (carbachol) competition assay, the proportions of HA- and LA-binding sites and the corresponding affinity con- stants of the MAChR were determined by using different concentrations of car- bachol. There were no significant changes in the proportions of HA- and LA-binding sites or the corresponding affinity constants for any of the treated groups, compared with their controls (Table II).

Discussion

The present investigation shows significant behavioural deviations both in mice treated neonatally with DDT and receiving bioallethrin as adults and in mice receiv- ing the vehicle as neonates and bioallethrin as adults. However, the spontaneous behaviour reactions developed in mutually opposite directions. Another finding was that adult exposure to bioallethrin increased the density of MAChR in the cerebral cortex in mice neonatally exposed to DDT from 70% (DDT + vehicle) to 90% (DDT + bioallethrin) of the level found in mice exposed to the vehicle both as neonates and adults. In mice receiving the vehicle neonatally and bioallethrin as adults no significant change in MAChR was noted. This indicates an increased susceptibility of the cholinergic system in adult mice neonatally exposed to DDT. In-

TABLE I

EFFECTS OF NEONATAL EXPOSURE TO DDT AND ADULT EXPOSURE TO BIOALLETHRIN ON THE MUSCARINIC RECEPTORS IN THE CEREBRAL CORTEX OF ADULT MICE a

Ten-day-old NMRI mice received a single dose of DDT (0.5 mg) or the vehicle (10 ml) per kg body wt. per os. At the age of 5 months they received bioallethrin (0.7 mg) or the vehicle (10 ml) per kg body wt. per os once daily for 7 days and were killed 72 h after the last administration. [3H]QNB binding (pmol/g protein, mean ± SD, (n)) was assessed in the P2 fraction (24). The statistical difference vs. the correspon- ding control was determined by Student's t-test.

Treatment at Cortex (n)

10 Days 5 Months

Control Control 902 4- 133 (8) Control Bioallethrin 895 ± 138 (13) DDT Control 628 ± 204 a (6) DDT Bioallethrin 812 ± 133 b (11)

ap = 0.039 compared with control (control-control). bp = 0.020 compared with control (DDT-control).

27

TABLE II

EFFECTS OF NEONATAL EXPOSURE TO DDT AND ADULT EXPOSURE TO BIOALLETHRIN ON HIGH- AND LOW-AFFINITY MUSCARINIC BINDING SITES (%) AND AFFINITY CONSTANTS (k) IN THE CEREBRAL CORTEX OF ADULT MICE

Ten-day-old NMRI mice received a single dose of DDT (0.5 rag) or the vehicle (10 ml) per kg body wt. per os. At the age of 5 months they received bioallethrin (0.7 rag) or the vehicle (10 ml) per kg body wt. per os once daily for 7 days and were killed 72 h after the last administration. The displacement study was performed on the P2 fraction (24). The binding parameters were estimated from [3H]QNB/carbachoi competition curves. The percentage values are means 4- SD (n) and the affinity constants are geometric means. The statistical evaluation between controls and treated mice of the percentage values and the affinity constants were made with Student's t-test and the Mann-Whitney U-test, respectively. All P-values exceeded 0.1.

Treatment at High-affinity site Low-affinity site

10 Days 5 Months % (n) k(~M) % (n) k(#M)

Control Control 14.9 4- 6.4 2.85 85.1 4- 6.4 272 (6) (6)

Control Bioallethrin 11.5 4- 4.0 0.84 88.5 ± 4.0 285 (8) (8)

DDT Control 10.1 ± 3.7 1.19 89.9 4- 3.7 342 (5) (5)

DDT Bioallethrin 14.7 4- 12.2 1.21 85.3 4- 12.2 323

(8) (8)

volvement of other transmitter systems cannot be excluded since structurally the MAChR are similar to the serotonin, norepinephrine and dopamine receptors [39-41] which can all act via G-proteins and thereby affect a variety of cellular responses [42]. Further, this study confirms previous results showing that neonatal exposure to DDT (0.5 mg/kg body wt.) leads to a hyperactive condition in adult mice and a decrease in the MAChR in the cerebral cortex not accompanied by any signifi- cant change in the subpopulations of MAChR [26,28].

Our data indicate that neonatal exposure to a toxicant, DDT, during neonatal life affects the reaction of the adult mouse to another agent, bioallethrin, which has a mechanism of action on nerve membrane sodium channels similar to that of the original compound (DDT) [10-14]. In earlier experiments we have seen that DDT and bioallethrin cause the same changes in cholinergic receptor variables and in behaviour in both neonatal and adult mice following neonatal exposure to these substances [24,25,27,28]. These results demonstrated that neonatal exposure to DDT and bioallethrin leads to an increase in the density of MAChR in the neonatal mice and a decrease in adult mice and a hyperactive condition in the adult mice. Regard- ing the change in MAChR, in both neonates and adults, there are interesting similarities and dissimilarities following bioallethrin treatment of neonatal mice, compared with bioallethrin treatment of adult mice neonatally exposed to DDT. Administration of bioallethrin for 7 days to adult mice, neonatally exposed to DDT, caused an increase in the density of MAChR, a result also found in neonatal mice

28

exposed to bioallethrin for 7 days. However, the increase in MAChR in the adult mice was not accompanied by any significant change in the proportion of HA- and LA-binding sites as observed in the neonatal mouse [25]. In mice neonatally receiv- ing the vehicle, followed by bioallethrin at the age of 5 months, no significant change in MAChR was observed. Thus, neonatal exposure to DDT appears to increase and/or modulate the reaction to bioallethrin in adult life. One explanation offered is that the change in proportions of HA- and LA-binding sites in the neonatal mice is a compensatory mechanism whose function is to copy the altered ACh turnover caused by an increased neuronal activity due to the bioallethrin treatment [25]. Generally speaking, changes in receptors are usually inverse to the changes in stimulation, thereby resulting in a pattern consistent with compensatory adaptation to the change in stimulation [43]. That the proportions of HA- and LA-binding sites were not altered in the adult brain might mean that the cholinergic system is affected and therefore unable to effect the compensatory modification of the muscarinic cholinergic neurotransmission. According to our previous data [24,25,27,28] and present results, there appears to be a time-related change in MAChR in the cerebral cortex following neonatal exposure to DDT or bioallethrin, as in the neonatal mice both treatments caused an increase in MAChR in the neonatal mice while with advancing age the amount of MAChR decreased, in comparison with the control animals. An age-related decrease in MAChR is known to occur [44,45] and ageing and cholinergic dysfunction have long been mutually associated [23]. Whether or not the increased MAChR density observed in bioallethrin-exposed mice, exposed neonatally to DDT, can in turn accelerate age-dependent changes in cholinergic receptors, is therefore of particular interest and remains to be investigated.

The behavioural aberration observed in mice exposed to DDT neonatally and as adults to bioallethrin, indicated a rearing pattern opposite all other treatments. Although this behavioural variable was attenuated, compared to mice neonatally exposed to DDT and the vehicle as adults, the rearing behaviour seemed to increase during the observational period. Rearing is often associated with explorative behaviour and the cause and persistence of this reaction in these mice remains to be determined in further studies.

In conclusion, the present study indicates that animals exposed as neonates to a persistent environmental hazard, DDT, react differently when again exposed as adults to a similar neurotoxic agent, than do animals neonatally exposed to vehicle only. The amount of DDT given in our studies was of physiological significance and toxicological relevance, since it is of the same order of magnitude as that to which animals and humans can be exposed during the lactation period [46-48].

Acknowledgements

The authors thank Dr. Tessier, Rousell Uclaf, France, for the gift of bioallethrin and Miss Anna Pettersson for excellent technical assistance. This work was finan- cially supported by grants from the Swedish Environmental Protection Board, The Swedish Work Environment Fund and the Bank of Sweden Tercentenary Foun- dation.

29

References

l E.H. Dustman and L.F. Stickel, The occurrence and significance of pesticide residues in wild animals. Ann. NY Acad. Sci., 160 (1969) 162.

2 C.A. Edwards, Persistent pesticides in the environment. CRC Monoseience Series, Chemical Rubber Co., Cleveland, 1970.

3 G. Woodard, R.R. Ofner and C.M. Montgomery, Accumulation of DDT in the body fat and its ap- pearance in the milk of dogs. Science, 102 (1945) 177.

4 D.E. Woolley and G.M. Talens, Distribution of DDT, DDD, and DDE in tissues of neonatal rats and in milk and other tissues of mother rats chronically exposed to DDT. Toxicol. Appl. Phar- macol., 18 (1971) 907.

5 M. Elliott, Synthetic insecticides designed from natural pyrethrins. Pontif. Accad. Sci. SCr. Varia., 41 (1977) 157.

6 J.E. Casida, D.W. Gammon, A.H. Glickman and L.J. Lawrence, Mechanisms of selective action of pyrethroid insecticides. Annu. Rev. Pharmacol. Toxicol., 23 (1983) 413.

7 W.N. Aldridge, An assessment of the toxicological properties of pyrethroids and their neurotoxicity. Crit. Rev. Toxicol., 21 (1990) 89.

8 D.W. Gammon, M.A. Brown and J.E. Casida, Two classes of pyrethroid action in the cockroach. Pestic. Biochem. Physiol., 15 (1981) 181.

9 R.D. Verschoyle and W.N. Aldridge, Structure-activity relationships of some pyrethroids in rats. Arch. Toxicol., 45 (1980) 325.

10 W. Wouters and J. van den Bercken, Action of pyrethroids. Gen. Pharmacol., 9 (1978) 387. 11 J. van den Bercken, L.M.A. Akkermans and J.M. van der Zalm, DDT-like action of allethrin in the

sensory nervous system of Xenopus laevis. Eur. J. Pharmacol., 21 (1973) 95. 12 T. Narahashi, Cellular and molecular mechanisms of action of insecticides: Neurophysiological

approach. Neurobehav. Toxicol. Teratol., 4 (1982) 753. 13 T. Narahashi, Nerve membrane ionic channels as the primary target of pyrethroids. Neurotox-

icology, 6 (1985) 3. 14 A.E. Lund and T. Narahashi, Kinetics of sodium-channel modification as the basis for the variation

in the nerve membrane effects of pyrethroids and DDT analogs. Pestic. Biochem. Physiol., 20 (1983) 203.

15 A.N. Davison and J. Dobbing, Applied Neurochemistry, Blackwell, Oxford, 1968, pp. 178-221, 253-316..

16 J.T. Coyle and H.I. Yamamura, Neurochemical aspects of the ontogenesis of cholinergic neurons in the rat brain. Brain Res., 118 (1976)429.

17 Y. Falkeborn, C. Larsson, A. Nordberg and P. Slanina, A comparison of the regional ontogenesis of nicotine- and muscarine-like binding sites in the mouse brain. Int. J. Dev. Neurochem., 1 (1983) 187.

18 M. Marchi, A. Cavigiia, P. Paudice and M. Raiteri, Calcium-dependent [3H]acetylcholine release and muscarinic autoreceptors in rat cortical synaptosomes during development. Neurochem. Res., 8 (1983) 621.

19 C.C. H6hmann and F.F. Ebner, Development of cholinergic markers in mouse forebrain. I. Choline acetyltransferase enzyme activity and acetylcholineesterase histochemistry. Dev. Brain Res., 23 (1985) 225.

20 C.C. H6hmann, C.C. Pert and F.F. Ebner, Development of cholinergic markers in mouse forebrain. II. Muscarinic receptor binding in cortex. Dev. Brain Res., 23 (1985) 243.

21 A.G. Karczmar, Cholinergic influences on behavior, in P.G. Waser (Ed.), Cholinergic Mechanisms, Raven Press, New York, 1975, pp. 501-529.

22 R.T. Bartus and H. R. Johnson, Short-term memory in the rhesus monkey: Disruption from the anti- cholinergic scopolamine. Pharmacol. Biochem. Behav., 5 (t976) 39.

23 R.T. Bartus, R.L. Dean III, B. Beer and A.S. Lippa, The cholinergic hypothesis of geriatric memory dysfunction. Science, 217 (1982) 1408.

24 P. Eriksson and A. Nordberg, The effects of DDT, DDOH-palmitic acid and a chlorinated paraffin on muscarinic receptors and th 0 sodium-dependent choline uptake in the central nervous system of immature mice. Toxicol. Appl. Pharmacol., 85 (1986) 121.

30

25 P. Eriksson and A. Nordberg, Effects of two pyrethroids, bioallethrin and deltamethrin, on sub- populations of muscarinic and nicotinic receptors in the neonatal mouse brain. Toxicol. Appl. Phar- macol., 102 (1990) 456.

26 P. Eriksson, L. Nilsson-H~kansson, A. Nordberg, A. Aspberg and A. Fredriksson, Neonatal ex- posure to DDT and its fatty acid conjugate - - Effects of cholinergic and behavioural variables in the adult mouse. Neurotoxicology, 11 (1990) 345.

27 P. Eriksson and A. Fredriksson, Neurotoxic effects of two different pyrethroids, bioallethrin and deltamethrin, on immature and adult mice: Changes in behavioural and muscarinic receptor variables. Toxcol. Appl. Pharmacol., 108 (1991) 78.

28 P. Eriksson, J. Ahlbom and A. Fredriksson, Exposure to DDT during a defined period in the neonatal life induces permanent changes in brain muscarinic receptors and behaviour in adult mice. Brain Res., 582 (1992) 277.

29 P. Eriksson, U. Lundkvist and A. Fredriksson, Neonatal exposure to 3,3 ',4,4 '-tetrachlorobiphenyl: changes in spontaneous behaviour and cholinergic muscarinic receptors in the adult mouse. Tox- icology, 69 (1991) 27.

30 A. Nordberg, X. Zhang, A. Fredriksson and P. Eriksson, Neonatal exposure induces permanent changes in brain nicotinic receptors and behaviour in adult mice. Dev. Brain Res., 63 (1991) 201.

31 V.H. Denenberg, Some statistical and experimental considerations in the use of the analysis-of- variance procedure. Am. J. Physiol., 246 (1984) R403.

32 T. Archer, A. Fredriksson, T. Lewander and U. S6derberg, Marble burying and spontaneous motor activity in mice: Interactions over days and the effect of diazepam. Scand. J. Psychol., 28 (1987) 242.

33 E.G. Gray and V.P. Whittaker, The isolation of nerve endings from brain: An electron-microscopic study of cell fragments derived by homogenization and centrifugation. J. Anat., 96 (1962) 79.

34 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193 (1951) 265.

35 A. Nordberg and B. Winblad, Cholinergic receptors in human hippocampus - - regional distribution and variance with age. Life Sci., 29 (1981) 1937.

36 N.J.M. BirdsaU, A.S.V. Burgen and E.C. Hulme, The binding of agonists to brain muscarinic recep- tors. Mol. Pharmacol., 14 (1978) 723.

37 A. Nordberg and G. Wahlstr6m, Changes in populations of cholinergic binding sites in brain after chronic exposure to barbital in rats. Brain Res., 246 (1982) 105.

38 R.E. Kirk, Experimental Design: Procedures for the Behavioural Science, Brooks/Cole, Belmont, CA, 1968.

39 J.R. Bunzow, H.H.M. Van Tol, D.K. Grandy, P. Albert, J. Salon, M. Christie, C.A. Machida and O. Civelli, Cloning and expression of a rat D2 dopamine receptor. Nature, 336 0988) 783.

40 R.A.F. Dixon, B.K. Kobilka, D.J. Strader, J.L. Benovic, H.G. Dohlman, T. Frielle, M.A. Bolanowski, C.D. Bennett, E. Rands, R.E. Diehl, R.A. Mumford, E.E. Slater, I.S. Sigal, M.G. Caron, R.J. Lefk0witz, C.D. Strader, Cloning of a gene and cDNA for mammalian beta-adrenergic receptor homology with rhodopsin. Science, 321 (1986) 75.

41 T. Kubo, F. Kazuhiko, A. Mikami, A. Maceda, H. Takahaski, M. Mishina, T. Haga, K. Haga, A. Ichiyama, K. Kanagara, M. Kojima, H. Matsuo, T. Hirose and S. Numa, Cloning, sequencing and expression of complementary DNA encoding the muscarinic acetylcholine receptor. Nature, 323 (1986) 411.

42 A.M. Speigel, Signal transduction by guanine nucleotide binding proteins. Mol. Cell. Endocrinol., 49 (1987) 1.

43 I. Creese and D.R. Sibley, Receptor adaptations to centrally acting drugs. Annu. Rev. Pharmacol. Toxicol., 21 (1981) 357.

44 A. Nordberg, The aging of cholinergic synapses: ontogenesis of cholinergic receptors, in I. Hanin (Ed.), Dynamics of Cholinergic Function, Advances in Behavioural Biology, Raven Press, New York, 1968, pp. 165-175.

45 A. Biegon, R. Duvdevani, V. Greenberger and M. Segal, Aging and brain cholinergic muscarinic receptors: an autoradiographic study in the rat. J. Neurochem., 51 (1988) 1381.

46 A. Bevenue, The 'bioconcentration' aspects of DDT in the environment. Residue Rev., 61 (1976) 37. 47 World Health Organisation (WHO), DDT and its derivatives. Environ. Health Criteria, 9 (1979) 57. 48 S.A. Slorach and R. Vaz, Assessment of human exposure to selected organochlorine compounds

through biological monitoring, Swed. Nat. Food Adm., Uppsala, 1983, pp. 134.