The developing cholinergic system as target for environmental toxicants, nicotine and polychlorinated biphenyls (PCBs): Implications for neurotoxicological processes in mice

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The Developing Cholinergic System as Target for Environmental Toxicants, Nicotine and Polychlorinated Biphenyls (PCBs): Implications for Neurotoxicological Processes in Mice PER ERIKSSON*, EMMA ANKARBERG, HENRIK VIBERG and ANDERS FREDRIKSSON

Department of Environmental Toxicology Uppsala University Norbyvi~gen 18 A S-752 36 Uppsala Sweden

(Received October 27, 1999; In final form February 24, 2000)

During neonatal life, offspring can be affected by toxic agents either by transfer via mother's milk or by direct exposure. In many mammalian species the perinatal period is characterized by a rapid development of the brain - "the brain growth spurt" (BGS). This period in the development of the mammalian brain is associated with numerous biochemical changes that transform the feto-neonatal brain into that of the mature adult. In rodents, the cholinergic transmitter system undergoes a rapid development during the neonatal period, a time when spontaneous motor behaviour also reaches peak activity. We have observed that low-dose exposure to environmental toxicants such as nicotine, polychlorinated biphenyls (PCBs) and polybrominated diphe- nylethers (PBDE, flame retardants) during the "BGS" can lead to irreversible changes in adult brain function in the mouse. The induction of persistent effects on behaviour and cholinergic nicotinic receptors in the adult animal appears to be limited to a short period during neonatal development. Furthermore, the neurotoxic effects were shown to develop over time, indicating a time- respons/time-dependent effect. This indicates that environmental toxicants, such as nicotine, PCBs and probably PBDEs, might be involved in the slow, implacable induction of neurodegenerative disorders and/or interfere with normal aging processes.

I N T R O D U C T I O N

Whether exposure to environmental agents can contribute to neuronal disorders is of special interest. The development of neurodegenerative disorders appears to be a complex interaction between genetic and environmental factors, e.g. the development of Alzheimer's disease (AD) and Parkinson's disease (PD) (James and Nord- berg, 1995). Neurodegenerative disorders such as AD are characterized by the impairments of memory and cognitive functions. The cholinergic system in particular has been implicated as an important role in aging and memory defect disorders. Dysfunctions of the cholinergic system have been shown to cause learning and memory impairment (Bartus et al., 1982; Over- street and Russell, 1991). Although AD is a disorder involving several neurotransmitter systems, the cholinergic system is severely and consistently affected (for review, see Whitehouse and Au, 1986). In normal aging there is usually a

* Correspondence to: Per Eriksson, Tel: +46 18 4712623, Fax: +46 18 518843, E-mail: Per.Eriksson@ebc. uu. se

37

38 PER ERIKSSON et al.

change and /or decrease in cholinergic receptors (Nordberg and Winblad, 1986; Nordberg et al., 1992; Narang, 1995).

Vulnerable periods during brain development

Mammalian development includes periods which can be critical for normal maturation. As the CNS develops, every region of and structure in the brain follows an intricately planned and precisely timed developmental sequence, for example neurogenesis, differentiation and synaptogenesis. Vulnerable periods during ontogenesis of the CNS can nevertheless be divided into two major courses of events. The first includes early brain development, a period during which the brain acquires its general adult shape and when the spongioblasts and neurob- lasts, precursors of glia cells and neurons, respectively, proliferate (Rodier, 1980; Rogers and Kavlock, 1996). Interference by xenobiotics during this period can cause malformation of the brain (Rodier et al., 1996; Rogers and Kavlock, 1996). The second period coincides with the brain growth spurt (BGS). It is then that the brain undergoes a series of rapid fundamental developmental changes, including the maturation of axonal and dendritic outgrowth; establishment of neural connections; synaptogenesis; cell, axon and dendrite death; and the proliferation of glia cells with accompanying myelinization (Davison and Dobbing, 1968; Kolb and Whishaw, 1989). These cytoarchitectural changes are accompa- nied by a vast number of biochemical changes that transform the feto-neonatal brain into that of the mature adult. This is also the stage of development when animals acquire many new motor and sensory faculties (Bolles and Woods, 1964), including advances in spontaneous motor behaviour (Campbell et al., 1969).

The BGS does not take place at the same time point in all mammalian species. In the human, this period begins during the third trimester of pregnancy and continues throughout the first 2

years of life. In mouse and rat the BGS is neonatal, spanning the first 3-4 weeks of life. One of the major neurotransmitters in the CNS is acetylcholine (ACh), which acts as the transmitter in the cholinergic pathways. In rodents, this transmitter system in the CNS undergoes rapid development during the first 3-4 weeks after birth (Coyle and Yamamura, 1976; Fiedler et al., 1987) when gradually increasing numbers of muscarinic and nicotinic receptors appear in the cerebral cortex and hippocampus (Kuhar et al., 1980; Falkeborn et aL, 1983; Slotkin et al., 1987; Fiedler et al., 1987). The cholinergic transmitter system is involved in many behavioural phe- nomena (Karczmar, 1975) and is closely related to cognitive functions (Drachman, 1977; Bartus et al., 1982).

Our previous investigations have shown that low-dose neonatal exposure to both persistent and non-persistent environmental agents can lead to disruption of brain function in the adult animal. Among the agents known to induce such neurotoxic effects are DDT (Eriksson et al., 1992; Eriksson, 1992), pyrethroids (Eriksson and Fre- driksson, 1991; Eriksson, 1992; Ahlbom et al., 1994), organophosphate (Ahlbom et al., 1995), nicotine (Nordberg et al., 1991), paraquat and MPTP (Fredriksson et al., 1993) and some polychlorinated biphenyls (Eriksson, 1998). The induction of behavioural and cholinergic disturbances that appears in adult animals has been shown in several cases to be limited to a short period during neonatal development, namely around postnatal day 10 (Eriksson et al., 1992; Ahlbom, et al., 1995; Eriksson, 1997; 1998; Eriks- son et al., 2000). Those studies also showed that these disturbances are induced at dosages that apparently have no permanent effect when administered to the adult animal. Exposure during this phase of development has also been shown to lead to an increased susceptibility to toxic agents at adult age, indicating that neonatal exposure to toxic agents can potentiate and /or modify the reaction to adult exposure to xenobiotics (Johansson et al., 1995; Talts et al., 1998; Eriksson and Talts, 1999).

CHOL1NERG1C ONTOGENY AND TOXICANTS 39

Aging

During aging, several transmitter systems undergo decreases in receptor number and function. Neurotransmitter receptor plasticity has also been shown to be impaired in the aged brain (Pilch and M6ller, 1988), leading to a reduced ability to adjust to changes in the environment (reviewed by Pedigo, Jr., 1994). Impaired respon- siveness of [~-adrenergic receptors has been reported (Greenberg and Weiss, 1978), and serotonin, dopamine and opiate receptors all appear to become more sparse with advancing age (Wong et al., 1984; Messing et al., 1980). Aging is also associated with progressive deterioration in learning and memory capability. As dysfunction in the cholinergic system has been shown to impair learning and memory (Bartus et al., 1982), it has been suggested that this system in particular plays an important role in the process of aging and in memory defect disorders, e.g. Alzheimer's disease (James and Nordberg, 1995; Nordberg et al., 1992, Whitehouse and Au, 1986).

Most animals have a distinct pattern of aging in the brain, which is subject to genetic control. Nonetheless, the neurobiological effects of aging display substantial individual variations (Finch, 1994). Several studies have suggested that varia- bility in learning and memory functions is a prominent feature of aging in the rodent. In these investigations, aged rats displayed a broad spread in learning capacity in a spatial learning test of Morris water-maze type, when compared with younger animals (reviewed in Rapp and Amaral, 1992).

The suspicion that environmental agents may interfere with the aging process or contribute to neuronal disorders is an intriguing issue. Since aging is a gradual and highly individual process, even careful studies might overlook potential low exposure levels responsible for only minor acceleration of the aging process.

Animal Model

It is often difficult to distinguish the effects of exposure in utero from effects due to breast milk

exposure alone, especially exposure from envi- ronmetal chemicals that are highly persistent, such as PCBs, where the exposure via the milk far exceeds the contribution made by maternal-fetal transfer (for ref., see Gallenberg and Vodicnik, 1989).

In the mouse the "brain growth spurt" ("BGS") occurs neonatally. By using the mouse as an animal model we can study the effect of a single toxicant administered directly to animals during different stages of the "BGS". Description of neurochemical and neuroreceptor analysis and behavioural methods are presented in the sepa- rate papers (e.g. see Fredriksson, 1994; Eriksson and Fredriksson 1996a,b, Eriksson 1998).

RESULTS AND DISCUSSION

Neonatal exposure to nicotine and its implication on cholinergic receptors and behaviour

Nicotine makes its impact on human health as a component of tobacco products. Underweight newborns, high rates of perinatal morbidity, mortality and Sudden Infant Death Syndrome, and persistent defects in learning and behaviour may all be associated with maternal smoking (see Slotkin, 1998). Animal studies show that prenatal exposure to nicotine (6mg/kg /day ) can lead to behavioural changes such as hyperactivity in the offspring (Tizabi et al., 1997). Long-term changes in cognitive function, together with effects on the noradrenergic system, have also been reported in adult rats exposed to nicotine during gestation. Nicotine is one of the most commonly used addictive sub- stances (Henningfield and Woodson, 1988). At least two major classes of brain nicotinic receptors in the vertebrate brain have been characterized, using radiolabelled ligands: those having high affinity for [3H]nicotine and [3H]acetylcholine, and those with high affinity for [125I]bungarotoxin (Nordberg, 1993; Whiting and

40 PER ERIKSSON et aI.

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Treatment age: lO-day-o ld B e h a v i o u r : 4 - m o n t h - o l d

Treatment age: 1 9 - d a y - o l d B e h a v i o u r : 4 - m o n t h - o l d

C C C N N N C C O N N N C C C N N N S L H S L H S L H S L H S L H S L H

6 0 - 8 0 6 0 - 8 0 6 0 - 8 0 TIME (rain) TIME (rnin) TIME (rain}

FIGURE 1 Neonatal mice at an age of either 3, 10, or 19 days were exposed to nicotine, 66 lag nicotine-base/kg b.wt. s.c. twice daily, on 5 consecutive days. Controls received 10 mI 0.9% NaCl/kg b.wt. s.c. The treated groups contained mice from 3-4 different litters. At adult age of 4 months, male mice were observed for spontaneous and nicotine-induced behaviour. The results from the spontaneous motor behavioural test variable "locomotion" indicated no change during a 60-rain observational period. The nicotine-induced behaviour was studied by using two different doses of nicotine, 40 and 80 lag/kg b.wt. s.c., and 10 ml 0.9% NaC1/kg b.wt. s.c. For statistical evaluation, ANOVA with split-plot design was used (Kirk, 1968). There were significant group x period interactions for "locomotion" in the 3-day-olds: [F(10,60)= 16.56], 10-day-olds: [F(10,60)-42.52], 19-day-olds: IF(10,60) - 19.25]. Pairwise testing between nicotine-injected (40 and 80 lag) and saline2injected mice was performed with the Tukey HSD test (c~=0.01) (Kirk, 1968). The different treatments are indicated by: N, nicotine (66 lag);,C, control; S, saline; L, nicotine 40 lag; H, nicotine 80 lag; and the statistical difference vs. saline is indicated by the asterisks p _< 0.01. [Data taken from Eriksson et aI., 2000]

Lindstrom, 1988; Wonnacott, 1986). Low-affinity nicotine-binding sites, also present in the human and rodent brain, resemble o~-bungarotoxin-binding sites (Nordberg, 1993; Nordberg et al., 1988; Wonnacott, 1986).

Neonatal exposure to low doses of nicotine (0.2 mg nicotine/kg body wt) have been shown to affect tlae nicotinic acetylcholine receptors (NAChR) in the neonatal mouse brain, leading to permanent disorder of brain function of adult mice, revealed as changes in behaviour and in binding properties of nicotinic receptors (Nord- berg et al., 1991). In a recent investigation we have shown that induction of these disturbances in the mouse seems to be limited to a short period of time during neonatal development. Neonatal mice at an age of either 3, 10, or 19 days were exposed to 66 gg nicotine-base/kg b.wt. s.c. twice daily for 5 consecutive days, and from cor- responding controls that received only the vehi-

cle, 0.9% NaC1. At an age of 4 months mice were observed for spontaneous behaviour and nicotine-induced behaviour. In the nicotine-induced behaviour test, low doses of nicotine are known to cause increased motoric activity in adult animals, whereas high doses can cause decreased activity (Nordberg and Bergh, 1985). Nicotinic receptor binding sites in the cerebral cortex were analysed by using tritium-labelled nicotine in a competition binding assay (Nordberg et al., 1991). This study showed that the spontaneous motor behaviour was not affected at the adult age of 4 months, however, the altered spontaneous behavioural response to nicotine was observed only in mice given nicotine between days 10 and 14 (Fig. 1) (Eriksson et al., 2000). Furthermore, this study showed also that even though the results of nicotine treatment was that low affinity (LA) binding sites could not be found during the neonatal period, the persist-

CHOLINERGIC ONTOGENY AND TOXICANTS 41

ence of this effect up to an adult age of 4 months was seen only in mice that were exposed on days 10 to 14 (Table I).

The persistent effects caused by exposure on days 10-14 appear not to be related to differences in uptake and/or retention of nicotine in this age group, compared with the other two age groups, as the nicotine exposure in all treatment groups caused a lack of LA binding sites during the neonatal period. Earlier studies have demonstrated dynamic changes in nicotinic receptors during development and maturation of the brain. Specific binding of [3H]nicotine has been detected in mouse brain during the late prenatal period. At birth there is a drop in [3H]nicotine binding sites for then increase during a period of 4 weeks when the adult level is reached (Zhang et al., 1990). With increasing age, from I month to 14 months in the rat cerebral cortex, the [3H]nicotine binding is known to decrease. At birth, only high affinity (HA) binding sites can be detected in mouse cerebral cortex, but between postnatal days 5 and 17, LA nicotine-binding sites became detectable (Nordberg, 1993). Recent studies in rat have also revealed persistent effects on LA nicotine-binding sites in adults following neonatal exposure to nicotine (0.1 mg/kg b.wt., s.c. twice daily) (Miao et al., 1998).

Different studies indicate the complexity of the development of nAChR subunit gene expression at mRNA level. Expression of mRNA for a2, a3, a4, co7, f32 and 134 is evident in rodent brain during neonatal development have been shown in several recent studies (Dominguez-de-Toro et al., 1997; Liu et al., 1996; Miao et al., 1998; Shacka and Robinson, 1998; Winzer-Serhan and Leslie, 1997). It has been suggested that the predomi- nant nAChR subtype in rodent brain consists of a4 and ~32 subunits and binds to nicotinic ago- nists with high affinity (Flores et al., 1992; Whit- ing and Lindstrom 1988), while 0~7 subunit may be the main component of o~-BgT binding (Coyle and Yamamura, 1976; Orr-Urtreger et al., 1997; Samuel et al., 1997). Whether the changes in nAChR, seen after nicotine exposure, are linked

to changes in mRNA levels of different subtypes is not known. Miao et al. (Miao et al., 1998) found a persistent increase in 3H-nicotine-binding sites and a lack of LA binding sites in rat cerebral cortex after neonatal exposure to nicotine, but these changes could not be correlated to different mRNA levels of various nAChR subunits. This lack of correlation has also been reported in mouse after chronic exposure to nicotine (Marks et al., 1992). However, prenatal nicotine exposure (2 m g / k g / d a y ) in rat has been shown to transiently increase nAChR subunits a7, co4 and 132 mRNA in brain, the effect being most pronounced on postnatal day 14 (Shacka and Robin- son, 1998). A null mutation of the a7 subunit has disclosed the absence of high-affinity 125I-a-BgT sites, but the high-affinity nicotine sites in the brain were not detectably affected (Orr-Urtreger et al., 1997). It has been suggested that the expression of the oc 7 subunit may be correlated with differences in nicotine binding, nicotine-induced seizures, and nicotine preference (Miner and Collins, 1989; Stitzel et al., 1997), and low-affinity nicotine-binding sites resembled to oc-bungarotoxin binding sites (Nordberg, 1993; Nordberg et al., 1988; Wonnacott 1986). Further- more, a recent study has shown that adult mice neonatally exposed to nicotine (66 gg nicotine-base/kg b.wt.) between days 10-14, do not respond at all with any increased activity when challanged with nicotine (Ankarberg et al., 1998). That is why the recent observed effect on low-affinity nicotine-binding sites are of particular interest in defining the functional role of low-affinity nicotine sites, as adult mice lacking these sites showed quite the opposite behavioural response to nicotine, compared with controls (Eriksson et al., 2000).

The time window for induction of the persistent effects in neonatal mouse coincides in humans with a period starting with the third trimester and continuing for several months after birth. This low-dose exposure is relevant as it is also known that nicotine is transferred via mothers milk, and at concentrations higher than the maternal plasma nicotine level (DahlstrOm et al., 1990).


TABLE I Proportion of high- and low-affinity nicotinic binding sites (%) and their affinity constants (k) in the cerebral cortex of the adult mouse (4 month-old) exposed neonatally to low doses of nicotine, PCB 28 and PCB 52 a

Treatment (mg/kg b.wt.) (n) High-Affinity Site Low-Affinity Site

% k (nM) % k@M)

Experiment 1

Treatment days 3-7

Vehicle (3) 76.2 • 3.9 3.4 23.8 • 3.9 1.6

Nicotine (3) 86.9 • 6.6 13.1 13.1 • 1.0 2.0

Treatment days 10-14

Vehicle (3) 82.6 • 7.2 3.4 17.4 _+ 7.2 1.6

Nicotine (3) 100 • 3.0 8.0 -a) -a)

Treatment days 19-23

Vehicle (3) 90.0 + 3.3 7.7 10.0 _+ 3.3 5.2

Nicotine (3) 85.8 + 3.7 7.0 14.2 _+ 3.7 5.4

Experiment 2

Treatment day 10

Vehicle (4) 78.5 • 9.5 9.2 21.5 • 9.5 18.2

PCB 28, 3.6 (3) 78.3 • 2.8 7.9 22.6 _+ 4.3 7.4

PCB 52, 4.1 (4) 98.4 + 2.3 13.3 -a) -a)

aExperiment 1: Male NMRI mice received nicotine, 66 ~tg nicotine base/kg b.wt., s.c. twice daily, on 5 consecutive days, at an age of either 3, 10, or 19 days. Controls received 10 ml 0.9% NaC1/kg b.wt, s.c Experiment 2: Male NMRI mice received a single oral dose of either PCB 28 (2,4',4- trichlorobiphenyl), PCB 52 (2,2'5,5'-tetrachlorobiphenyl) or the 20% fat emulsion vehicle on day 10. The mice were killed at an adult age of about 6 months. The binding parameters were estimated from (3H)nicotine/(-)nicotine competition curves performed on P2 fractions pooled from 2 animals, as described earlier (Nordberg et al., 1991). The percentage values are means _+ SD and the affinity constants are geometric means. a) The evaluation of the goodness of fit of the material for each treatment to one- and two-site models, based on the "the extra sum of squares" principle (Draper and Smith, 1966, as cited by Munson and Rodbard, 1980) revealed that binding curves from mice treated with PCB 52 on day 10 and from mice treated with nicotine between days 10-14 could not be fitted to a two-site model. [Data taken from Eriksson and Fredriksson 1996a, and Eriksson et al., 2000.]

Neonatal exposure to ortho-substituted PCB congeners and its implications on cholinergic receptors and behaviour

P o l y c h l o r i n a t e d b i p h e n y l s (PCBs) h a v e b e e n

u s e d i n a w i d e v a r i e t y of c o m m e r c i a l a n d i n d u s -

t r ia l p r o d u c t s s u c h as h y d r a u l i c - a n d h e a t t r ans -

fer f l u id s , d i e l e c t r i c f l u i d s in c a p a c i t o r s a n d

t r a n s f o r m e r s , p l a s t i c i z e r s , l u b r i c a n t s a n d f l a m e

r e t a r d a n t s (see H u t z i n g e r e t al., 1974). T h e s e

p h y s i c a l a n d c h e m i c a l p r o p e r t i e s t h a t m a d e

P C B s i n d u s t r i a l l y u s e f u l a l so m a d e t h e m res i s t -

a n t to d e g r a d a t i o n a n d t h e i r u s e h a s t h e r e f o r e

r e s u l t e d in c o n t a m i n a t i o n o f t h e g l o b a l e n v i r o n -

m e n t . C o n s e q u e n t l y P C B s h a v e b e c o m e b i o c o n -

c e n t r a t e d in t h e f o o d c h a i n a n d a c c u m u l a t e

r e a d i l y in m a m m a l s .

H u m a n e p i d e m i o l o g i c a l s t u d i e s s u g g e s t t h a t

pe r • e x p o s u r e to P C B s c a n h a v e d e v e l o p -

m e n t a l n e u r o t o x i c e f fec t s (Fe in e t al., 1984; J a c o b -

s o n et al., 1990; J a c o b s o n a n d J a c o b s o n , 1996;

R o g a n e t al., 1988). E x p e r i m e n t a l s t u d i e s in an i -

m a l s h a v e s h o w n t h a t c o m m e r c i a l m i x t u r e s o f

P C B s c a n c a u s e b e h a v i o u r a l a b e r r a t i o n s a n d


changes in brain neurotransmitter metabolism (see Seegal and Shain, 1992; Seegal and Schantz, 1994; SeegaL 1996). Exposure of mice, rats and monkeys to commercial mixtures of PCBs during development has been shown to produce long-term neurobehavioural changes (for ref., see Tilson et al., 1990; Tilson and Harry, 1994).

In a series of reports we have shown that low-dose exposure of neonatal mice to PCBs can lead to disruption of adult brain function, and also to an increased susceptibility to toxic agents at adult ages (Eriksson, 1998). The results of the spontaneous motor behaviour tests have shown a dose-response related disruption of habituation in 4-month-old mice exposed to PCB 28 (2,4,4'-trichlorobiphenyl, 0.7-14 pmol/kg b.wt), PCB 52 (2,2',5,5'-tetrachlorobiphenyl, 0.7-14 ~tmol/kg b.wt.) (Fig. 2) or PCB 153 (2,2',4,4',5,5'-hexachlorobiphenyl, 0.7-14 pmol/kg b.wt.) on postnatal day 10 (Eriksson and Fredriksson, 1996a,b; Eriksson, 1998). Nor- mal habituation is defined here as a decrease in locomotion, rearing, and total activity variables in response to the diminished novelty of the test chamber over a 60-minute test period, divided into three 20-min periods. Habituation was normal in the control animals, but mice treated with the different PCBs were obviously hypoactive during the first part of the 60-min period, while toward the end of the test period they became hyperactive. These studies indicated also that the functional disorder does indeed worsen with increasing age (Eriksson, 1998). In mice exposed to PCB 153 the aberrations were obviously more pronounced in 4-month-old than in 2-month-old animals. The difference in the locomotor variable during the first 20-min spell of the 60-min observational period, between mice given the higher dose of PCB 153 (5.1 mg /kg b.wt.) and the controls, was about 45% in 4-month-olds, compared with about 32% in 2-month-olds. This time-dependent effect was even more pronounced during the last 20-min period.

The ability of adult mice to learn and remem- ber was studied using two different types of

maze, viz. a radial maze (PCB 28, PCB 52, PCB 153) and a swim maze of the Morris water-maze type (PCB 28, PCB 52). The maze performance tests revealed that mice exposed to the highest dose of PCB 52 (4.1 mg /kg b.wt.) and of PCB 153 (5.1 mg / kg b.wt.) performed significantly worse than control animals, at age 4 and 6 months, respectively. In the 8-arm maze the mice exposed to PCB 52 and PCB 153 displayed longer latency to aquire the pellets and those given PCB 52 also tendened to make more errors, indicating impaired working memory in these animals. This dysfunction too can worsen with age. In mice given PCB 153 the derange- ment was not evident in the 4-month-old animals, whereas 2 months later (in 6-mont-old) they were significantly worse than controls. In the swim maze, measuring spatial learning, there was a 4-day acquisition period followed by reversal trials on the fifth day, when the platform was moved. In control mice and those given PCB 28 or PCB 52, latency to locate the platform decreased during acquisition training, and all tested animals performed equally well at the end. However, in the the reversal trials, mice exposed to the highest dose of PCB 52 did not improve in finding the new location of the platform, as did control mice and those exposed to PCB 28 (0.36 and 3.6 mg /kg b.wt.) or PCB 52 (0.41mg/kg b.wt.) (Fig. 3) (Eriksson and Fre- driksson, 1996a). In mice exposed neonatally to PCB 52 (4.1 mg /kg b.wt.) and showing deficits in the learning and memory tests, the nicotinic cholinergic receptors were found to be affected (Table I). In these animals only the high affinity (HA) binding sites of nicotinic receptors were present in the cerebral cortex, whereas controls and mice exposed to PCB 28 (3.6 mg /kg b.wt.), both HA and low affinity (LA) binding sites were present in proportions of about 80% and 20%, respectively. These proportions of HA- and LA binding sites are in agreement with previ- ously reported proportions of nicotinic binding sites in adult mice (Nordberg et al., 1991; Eriks- son et al., 2000). No significant effect was


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FIGURE 2 Spontaneous behaviour of 4-month-old NMRI male mice exposed to a single oral dose of either PCB 28 (2,4,4'-trichlorobiphenyl), PCB 52 (2,2',5,5'-tetrachlorobiphenyl), or the 20% fat emulsion vehicle at a neonatal age of 10 days. Statistical analysis of behavioural data were submitted to ANOVA using a split-plot design (Kirk, 1968). Pairwise testing between PCB-exposed and control groups was performed with the Tukey HSD tests. The treatment groups are indicated by: 1) control, 10 ml fat emulsion vehicle/kg body weight; 2) PCB 28, 0.18 mg (0.7 I-tmol)/kg body weight; 3) PCB 28, 0.36 mg (1.4 ~mol)/kg body weight; 4) PCB 28, 3.6 mg (14 Hmol)/kg body weight; 5) PCB 52, 0.20 mg (0.7 ~tmol)/kg body weight; 6) PCB 52, 0.41 mg (1.4 Hmol)/kg body weight; 7) PCB 52, 4.1 mg (14 I~mol)/kgbody weight. The height of each bar represents the mean + SD of 8 animals. The statistical difference vs. control is indicated by P < 0.01. [Data taken from Eriksson and Fredriksson (1996a).]


observed on the density of muscarinic acetylcholine receptors in hippocampus, which indicates that nicotinic acetylcholine receptors may be affected in animals with deficits in memory and learning. The swim maze of the Morris water-maze type, with its submerged platform, is designed to measure spatial learning, which is suggested to be correlated with cholinergic function (Whishaw, 1985; Lindner and Schallert, 1988). Agonist and antagonist compounds active on both muscarinic and nicotinic receptors can affect the maze performance of rodents (Hodges et al., 1991). Taken together, the results from these two memory and learning tests revealed that mice exposed neonatally to the highest dose of PCB 52 (4.1 mg /kg b.wt.) experienced obvi- ous difficulty in tasks requiring new/relearning and working memory.

It has been suggested that the high affinity nicotinic receptors are mainly presynaptic, thereby affecting the release of and / or the content of acetylcholine, as well as of other neurotransmitters, such as dopamine and serotonin (Beani et al., 1989; Balfour, 1989; Westfall et al., 1989). How- ever, neonatal exposure to either PCB 28 or PCB 52 did not significantly affect the content of dopamine and its metabolites (3,4-dihydroxy- phenylacetic acid, homovanillic acid) or of serotonin and its metabolite (5-hydroxyindoleacetic acid) in striatum.

Whether the observed changes in spontaneous behaviour and in cholinergic nicotinic receptors in adult mice neonatally exposed to PCB 52 would include effects on the cholinergic and/or the dopaminergic system, the behavioural response of adult animals to a cholinergic (nicotine) and a dopaminergic (d-amphetamine) agent was investigated. Mice treated neonatally with PCB 52 showed the same non-habituating behaviour at the age of 4 months as earlier observed (Eriksson and Fredriksson, 1996a), but the response to low doses of nicotine was quite the reverse, compared with the controls. In control animals, hyperactivity was seen after 40 and 80 ~tg nicotine base, whereas the PCB 52-treated

mice were obviously hypoactive (Fig. 4) (Eriks- son and Fredriksson, 1996b). This response to nicotine in the PCB 52-treated mice is the same as we found earlier in mice treated neonatally with nicotine (Nordberg, et al., 1991; Eriksson et al., 2000). It was of particular interest that, as adults, these neonatally nicotine-treated animals lacked the LA nicotinic binding sites, as did the PCB 52-treated mice (Nordberg et al., 1991, Eriksson and Fredriksson, 1996a; Eriksson et al., 2000). Furthermore, neonatal exposure on postnatal day 10 coincide with the rapid development of the cholinergic system (Falkeborn et al., 1983; Fiedler et al., 1987), and in the mouse this time point in the development of nicotinic receptors occurs when the LA nicotinic binding sites become detectable (Nordberg, 1993).

D-amphetamine can be used as a tool to detect possible changes in the functioning of the dopaminergic system. D-amphetamine is known to increase spontaneous motor activity in rodents. Its action on the dopaminergic neurons is associated with a non calcium dependent increased release of dopamine from extravessic- ular stores and an inhibition of its re-uptake in dopaminergic neurons, thereby potentiating the CNS effect of dopamine. The responses to d-amphetamine in 4-month-old mice treated neonatally with PCB 52 or vehicle showed a significant dose-related increase in spontaneous motor activity. The two groups responded fairly similarly to d-amphetamine (Eriksson and Fre- driksson, 1996b). Although several studies have demonstrated that the dopaminergic system in adult animals is affected by acute, chronic, and developmental exposure to commercial mixtures of PCBs (see Seegal and Shain, 1992, Seegal and Schantz, 1994), and in vitro studies have shown lightly chlorinated ortho-substituted PCBs to be potent in reducing dopamine in PC 12 cells (Angus and Contreras, 1996; Seegal et al., 1990; Shain et al., 1991) and in bovine adrenal chroma- ffin cells (Messeri et al., 1997) the present exposure to PCB 52 or PCB 28 did not affect the levels of dopamine, serotonin or their metabolites in


28

24

2O

= 1 6 r ._J

12

8

4

0

Trials 1-5

* =control

A=PCB 52 (0.41 mg/kg body wt.) ~k, o =PCB 52 (4.1 rng/kg body wt.)

o

l i l l l l i l l - ' l l i l l l l l l l l [ ~ [ t i l l

Day 1 ~ ~ Day 2 ~ 4 - Day 3 ~ 4- - Day 4 ~ ~ Day 5

FIGURE 3 Swim-maze performance of 5-month-old NMRI male mice exposed to a single oral dose of either PCB 52 (2,2',5,5'-tetrachlorobiphenyl) or the 20% fat emulsion vehicle at a neonatal age of 10 days. Latencies to reach the platform were measured during acquisition (days 1-4) and during reversal trials (day 5). Statistical analysis: the behavioural data, day 1 to day 4, were submitted to ANOVA using a split-plot design (Kirk, 1968). Statistical analysis of the behavioural data for day 5 were submitted to paired t-test and ANOVA 1-way combined with Duncan's test. Days i to 4: All mice improved their ability to locate the platform and there were no significant group x period interactions for PCB 52 [F(6,99)=2.06] vs. controls. Day 5: Controls significantly improved their ability to find the new location [paired t-test trial i vs. trial 5, P_<0.01)]. Mice receiving PCB 52 (4.1 rng/kg body weight) differed significantly from controls [F(2,35)-3.9; Duncan, P _< 0.05]. The treatment groups are indicated by: *) control, 10 ml fat emulsion vehicle/kg body weight; G) PCB 52, 0.41 mg (1.4 ~mol)/kg body weight; o) PCB 52, 4.1 mg (14 ~tmol)/kg body weight. Each point represents the mean of 12 to 14 animals. [Data taken from Eriksson and Fredriksson 1996a.]

s t r i a t u m in a d u l t a n i m a l s (Er iksson a n d Fre-

d r i k s s o n , 1996 a). H o w e v e r , in o u r s t u d y the

s i m i l a r i t y o n d - a m p h e t a m i n e - i n d u c e d b e h a v -

i o u r b e t w e e n n e o n a t a l PCB 52 a n d veh ic le

t r e a t e d a n i m a l s is cons i s t en t w i t h the o b s e r v a -

t ion tha t d o p a m i n e leve ls w e r e no t a f fec ted b y

n e o n a t a l PCB 52 t r e a t m e n t (Er iksson a n d Fre -

d r i k s s o n , 1996a).

The i n d u c t i o n of b e h a v i o u r a l d i s t u r b a n c e s b y

n e o n a t a l e x p o s u r e to PCB in the m o u s e a lso

s e e m s to be l i m i t e d to a s h o r t p e r i o d of t ime d u r -

ing n e o n a t a l d e v e l o p m e n t , as ea r l i e r s een for nic-

ot ine. In mice e x p o s e d to PCB 52 ( m g / k g b .wt . ) a

s ign i f i can t b e h a v i o u r a l a b e r r a t i o n w a s o b s e r v e d

in a d u l t (4 m o n t h - o l d ) m ice g i v e n PCB 52 at a n

age of e i the r 3 or 10 d a y s (Er iksson, 1998). W i t h

r e g a r d to a p r o n o u n c e d r e t e n t i o n of PCB 52 in

the n e o n a t a l b r a i n the b e h a v i o u r a l d i s t u r b a n c e s

seen af ter n e o n a t a l e x p o s u r e on d a y 3 m i g h t be

a t t r i b u t a b l e to the a m o u n t of PCB 52 p r e s e n t o n

d a y 10 b e i n g e n o u g h to i n d u c e b e h a v i o u r a l d i s -

t u r b a n c e s (Er iksson 1998).


600

z 0 I-- 400 o I o I 0 200 I 0 ._]

[ ] Control [ ] PCB 52

S L H S L H ~ S L H S L H S L H S L H

6 0 - 8 0 8 0 - 1 0 0 1 0 0 - 1 2 0

TIME PERIODS {rnin)

FIGURE 4 Nicotine-induced behaviour of 4-month-old NMRI male mice given a single oral dose of PCB 52 (2,2',5,5'-tetrachlorobiphenyl, 4 . l ing (14 btmol)/kg body weight) or the 20% fat emulsion vehicle (10 ml /kg body weight) at a neonatal age of 10 days. Determination of spontaneous motor activity is described in Materials and meth- or The nicotine-induced behaviour was studied by using two different doses: 40 and 80 btg/kg body weight, s.c., and 10 ml 0.9% NaC1/kg body weight, s.c. Statistical evaluation ANOVA with a split-plot design was used (Kirk, 1968). There were significant group x period interactions [F(10,84) = 78.7, F(10,84) = 163.2, F(10,84) = 51.5], for the variables "locomotion", "rearing" and "total activity", respectively. Pairwise testing between nicotine-injected (40 and 80 bt g) and saline-injected mice was performed with Tukey HSD tests (Kirk, 1968). The different injections are indicated by: S) saline; L) nicotine 40 Ixg; H) nicotine 80 ~tg; and the, statistical difference vs. saline is indicated by asterisks, P _< 0.01. The height of each bar represents the mean value +SD of 8 mice. [Data taken from Eriksson and Fredriksson, 1996b]

Possible interactive effects between nicotine, PCBs and other environmental toxicants affecting the cholinergic system

In different studies we have seen that nicotine, present in tobacco products, and PCB, one of the most widely spread environmental toxicants, can affect the same type of cholinergic receptor in the cerebral cortex, namely the nicotinic receptors (Eriksson and Fredriksson, 1996a; Eriksson 1998; Eriksson et al., 2000; Nordberg et al., 1991). The effects of neonatal exposure to low doses of nicotine on spontaneous behaviour and nicotine-induced behaviour in 4-month-old mice and on the development of nicotinic receptors in the brain showed that nicotine can prevent the development of LA nicotinic binding sites in the

cerebral cortex and that this exposure induces a different behavioural response to nicotine in adult animals (Nordberg et al., 1991). This effect is similar to that observed after neonatal exposure to PCB 52 (Eriksson and Fredriksson, 1996a,b). In a recent study we observed that co-exposure to both nicotine and PCB 52 affects the development of nicotinic receptors, measured by binding of alpha-bungarotoxin. Alpha-bungarotoxin bound significantly less in mice neonatally exposed to both nicotine and PCB 52. This interactive effect was also evident in the spontaneous behaviour response to nicotine at adult age, where the neonatally co-exposed animals showed an increased response to nicotine (Ankarberg et al., 1998).

A new pollutant found in our environment and also in humans are certain brominated flame retardants. Polybrominated diphenyl ethers (PBDEs) are used in large quantities as flame-retardant additives in polymers, especially in the manufacture of a great variety of electrical appliances, including television and computer casing, building materials, and textiles (Environmental Health Criteria, 1994). One of the earliest reports of PBDE in our environment came in 1981 (Andersson and Blomkvist, 1981). PBDEs are persistent compounds that appear to have an environmental dispersion similar to that of PCB and DDT (Sellstr6m et al., 1993). PBDE has been found in various wildlife species and in human adipose tissue (Stanley et al., 1991). The PBDE congeners that dominate in environmental and human samples are 2,2',4,4'-tetrabro- modiphenylether (PBDE 47) and 2,2',4,4',5-pentabromodiphenylether (PBDE 99). These agents together with tetrabromo-bis-phe- nol-A (TBBPA) have been also detected recently in human plasma samples (Klasson-Wehler et al., 1997; Sj6din et al., 1999). The PBDEs are now also seen to increase in mother's milk (Noren and Meironyt6, 1998; Meironyt6 et al., 1998). In recent experiments we have seen that neonatal exposure to PBDE 47 and PBDE 99 can cause disturbances in spontaneous behaviour in a manner


similar to that described for PCBs (see Eriksson, 1998). Learning and memory in a swim maze of Morris water maze type was also found to be affected in the same manner as observed after neonatal exposure to PCB 52 and PCB 126 (see Eriksson, 1998), namely re-learning was significantly impaired. Another striking feature was that the effects on spontaneous behaviour

worsen with age, again as associated with PCB 52, PCB 153, PCB 126 and PCB 169 (see Eriksson, 1998). In order to explore whether the observed changes in spontaneous behaviour in adult mice neonatal ly exposed to PBDE 99 (8 m g / k g body weight) would include effects on the cholinergic system, the behavioural response of adult animals to a cholinergic (nicotine).agent was also studied. The response to a low dose of nicotine

was quite the reverse, compared with the controls. In control animals, hyperact ivi ty was seen after 80 gg nicotine base, whereas the PBDE 99-treated mice were obviously hypoactive. This response to nicotine in the PBDE 99-treated mice is the same as we found earlier in mice treated neonatal ly with PCB 52 (Eriksson, 1998) and nicotine (Nordberg et al., 1991; Eriksson et al., 2000). These observed similarities in behavioural disturbances related to cholineric system indicate that PBDEs can be a novel group of environ-

menatal neurotoxicants. Furthermore, the interactive effects be tween environmental toxicants affecting the cholinergic system call for future research.

CONCLUDING REMARKS

Our investigations have shown that low-dose exposure to nicotine and to certain PCBs during the per iod of rapid deve lopment of the neonatal

brain (so-called brain growth spurt) and cholinergic system, in the mouse, can give rise to irreversible changes in adult brain function.

Susceptibility to develop such irreversible disturbances can be limited to a defined develop-

mental phase of the brain growth spurt, and of the developing cholinergic system in the perinatal / neonatal brain.

The increased susceptibility to nicotine at adult age indicates that neonatal exposure to nicotine and to PCB can potentiate a n d / o r modify reactions to adult exposure to xenobiotics.

The disturbed spontaneous behaviour, and impaired learning and memo ry were shown to develop over time, indicating a t ime-response / t ime-dependent effect. This indi-

cate that environmental toxicants, such as nicotine, PCBs and probably PBDEs, might be involved in the slow, implacable induct ion of neurodegenerat ive disorders a n d / o r interfere with normal aging processes.

The recent findings that developmental expo-

sure PBDE can cause similarities in behavioural disturbances as seen for PCBs is of special interest, not only for PDBEs as a single agent but of possible interactive effects be tween these new environmental agents and other toxicants affecting the cholinergic system, such as nicotine and PCBs.

Acknowledgements This series of investigations has been suppor ted by grants f rom the Swedish Environmental Pro-

tection Board, the Bank of Sweden Tercentenary Foundation, the Swedish Council for Work Life Research, the Foundat ion for Strategic Environ- mental Research and the Nordic Council of Min- isters.

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The developing cholinergic system as target for environmental toxicants, nicotine and polychlorinated biphenyls (PCBs): Implications for neurotoxicological processes in mice