Effects of developmental co-exposure to methylmercury and

2,20,4,40,5,50-hexachlorobiphenyl (PCB153) on cholinergic

muscarinic receptors in rat brain

Teresa Coccini a,*, Giovanna Randine a, Anna F. Castoldi a, Philippe Grandjean b,Guido Ostendorp c, Birger Heinzow c, Luigi Manzo a,d

a IRCCS Salvatore Maugeri Foundation, Toxicology Division, Institute of Pavia, Pavia, Italyb University of Southern Denmark, Institute of Public Health, Department of Environmental Medicine, Odense C, Denmark

c Landesamt fur Gesundheit und Arbeitssicherheit des Landes Schleswig-Holstein, Department Environmental Health, Kiel, Germanyd University of Pavia, Department of Internal Medicine and Therapeutics, Toxicology Division, Pavia, Italy

Received 26 September 2005; accepted 23 December 2005

Available online 7 February 2006

Abstract

The developing nervous system is thought to be particularly sensitive to polychlorinated biphenyls (PCBs) present as food contaminants

together with methylmercury (MeHg). Effects of perinatal co-exposure to PCB153 and MeHg on brain cholinergic muscarinic receptors (MRs)

were investigated by saturation binding studies in mature and immature rats. MeHg alone (1 mg/kg/day, GD7–PND7) enhanced cerebral MRs

more in dams (87% and 60% in cerebellum and cerebral cortex, respectively) than in PND21 pups (0–50%) in accordance with the higher Hg levels

detected in the adult brain (7–9 mg/g) than in the male and female offspring’s brain (1.5–2.8 mg/g). Prenatal administration of PCB153 (20 mg/kg/

day, GD10–GD16), leading to higher contaminant levels in the offspring brain than in that of adults (25–66 mg/g versus 3 mg/g), induced cerebral

MR changes of similar extent at both ages, namely decreased cerebellar (20–30%) and increased cortical MR density (40–50%). Co-exposure to

PCB and MeHg had no more effect than exposure to either compound alone on cerebral cortex MRs, whereas, in the cerebellum, the combined

treatment induced a PCB-like lowering of the MR density that masked the MeHg-induced receptor increase. None of the treatments affected the

striatal and hippocampal MRs. A lower MeHg dose (0.5 mg/kg/day) was without any effect on cerebral MRs. These results show that MRs are one

of the sensitive biochemical endpoints of the central nervous system altered by developmental exposure to MeHg and PCB153. Cerebral cortex and

cerebellum were the most susceptible targets in the response to these neurotoxicants. MR changes were detected in both immature and adult

animals and the interaction of MeHg and PCB153 at the level of these receptors occurred in a non-additive manner.

# 2006 Elsevier Inc. All rights reserved.

Keywords: Neurotoxicity; Prenatal exposure; Lactation; PCB153; Mixture; Cerebral cortex; Striatum; Hippocampus; Cerebellum

NeuroToxicology 27 (2006) 468–477

1. Introduction

Methylmercury (MeHg) is a ubiquitous environmental

neurotoxicant particularly affecting brain development. It is

readily distributed throughout the body, easily penetrating the

blood–brain and placental barriers. The susceptibility of the

developing central nervous system (CNS) to MeHg is well

established according to epidemiological and experimental

evidence (NRC, 2000; USEPA, 2001). Neurodevelopmental

alterations have been reported in in utero MeHg-exposed

* Corresponding author. Tel.: +39 0382 592416; fax: +39 0382 24605.

E-mail address: tcoccini@fsm.it (T. Coccini).

0161-813X/$ – see front matter # 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.neuro.2005.12.004

children through a maternal diet rich in seafood, in the absence

of overt toxicity in the mothers. These effects resulted from an

average intakes below a previously defined threshold of

1 mg MeHg/kg bw/day that corresponded to 10 mg/g total Hg

in maternal hair (Grandjean, 1999; NRC, 2000; USEPA, 2001).

Polychlorinated biphenyls (PCBs) are also neurotoxic and

occur as contaminants in seafood together with MeHg (Weihe

et al., 1996). Neurobehavioural and neurochemical effects of

PCBs have been demonstrated by experimental investigations

in animals exposed during the pre- or early post-natal period

(Tilson and Kodavanti, 1997; Rice, 1999). In humans, increased

in utero exposures to PCBs have been associated with

neurodevelopmental abnormalities, including decreased intel-

ligence quotients (Jacobson and Jacobson, 1996). Attention has

T. Coccini et al. / NeuroToxicology 27 (2006) 468–477 469

therefore also been focused on the potential hazard resulting

from combined environmental exposures to low levels of PCBs

and MeHg (Grandjean et al., 2001; Longnecker et al., 2003).

Studies in the Faroe Islands have documented neurodevelop-

mental deficits in children whose mothers were exposed during

pregnancy, through seafood diet, to both MeHg and PCBs

(Grandjean et al., 1997; Weihe et al., 1996). While the PCB

exposure did not seem to explain or affect the MeHg-associated

neurotoxicity in the Faroese children (Budtz-Jørgensen et al.,

1999), PCB-associated effects appeared more prominent when

the MeHg exposure was high at the same time (Grandjean et al.,

2001). Because of the limited power of observational

epidemiological studies in disentangling possible interactions

of chemical mixtures, experimental studies using appropriate

animal models should be designed to determine the specific role

of MeHg or PCBs in producing neurodevelopmental effects. In

choosing the experimental system, attention must be paid to the

gender dependence of MeHg neurotoxicity (Gimenez-Llort

et al., 2001; Rossi et al., 1997).

A variety of PCB congeners occurs in seafood in association

with MeHg, and their neurotoxic profiles are still not well

defined (Bowers et al., 2004). 2,20,4,0,5,50-Hexachlorobiphenyl

(PCB153) is one of the most prevalent PCB congener of

environmental exposure (Longnecker et al., 2003) and also

constitutes about 25% of the PCB exposure in the Faroes

(Fangstrom et al., 2002). It is a di-ortho congener thought to

mediate its neurotoxicity via non-dioxin-like mechanisms.

Experimentally, either prenatal or lactational exposure to

PCB153 has produced long-lasting impairment in learning or

behaviour in female rats only (Holene et al., 1998; Schantz et al.,

1995). Long-term potentiation has also been found modified in

the developing rats following continuous gestational and

lactational exposure to this congener (Hussain et al., 2000).

The present study focused on interactive effects on the

developing cholinergic system of the brain. The cholinergic

system is essential for normal brain development as a

modulator of neuronal proliferation, migration and differentia-

tion processes (Hohmann and Berger-Sweeney, 1998) The

cholinergic muscarinic receptors (MRs), in particular, are

involved in several CNS functions, including learning and

memory (Levine et al., 2001). Several environmental com-

pounds that affect the cholinergic system have been also shown

to produce neurobehavioural alterations in the developing

organism (Tang et al., 2003). Recent gene-technology studies

using MR-deficient mice further support the critical role of

MRs in higher brain functions (Wess, 2003).

Substantial evidence indicates that the cholinergic muscari-

nic system can be affected by in vivo and in vitro exposure to

MeHg (Castoldi et al., 1996; Coccini et al., 2000; Eldefrawi

et al., 1977; Hrdina et al., 1976; Kobayashi et al., 1979; Limke

et al., 2004; Tsuzuki, 1981; Von Burg et al., 1980). The density

of cerebral MRs has been found to increase in a dose- and brain

area-dependent manner in adult female rats administered

repeatedly to low doses of MeHg (Coccini et al., 2000).

With regard to effects of PCBs on the cholinergic system,

limited data are available. Changes in hippocampal cholinergic

MR density have been shown to occur after administration of a

single dose of PCB77 to 10-day-old mice, 7 days as well as 4

months after treatment (Eriksson, 1988; Eriksson et al., 1991).

The present study aimed at evaluating the effects of the

perinatal exposure to MeHg (0.5 and 1 mg/kg bw/day, from

gestational day (GD7) to post-natal day (PND7) and PCB153

(20 mg/kg bw/day, from GD10 to GD16), alone and in

combination, on the density of MRs in selected brain areas of

weanling rat pups and, for comparison, on their dams. The MeHg

doses were chosen based on previous observations showing (i)

effects of MeHg on brain and lymphocyte MRs in adult female

rats at daily doses up to 2 mg/kg (Coccini et al., 2000) and (ii)

early and delayed neurochemical and behavioural alterations in

rats exposed to 0.5 mg MeHg/kg bw/day from GD7 to PND7

(Gimenez-Llort et al., 2001; Rossi et al., 1997). PCB153 protocol

of exposure was adopted on the basis of data showing learning

deficits in adult rats exposed prenatally to 16–64 mg PCB153/

kg/day from GD10 to GD16 (Schantz et al., 1995).

The specific aims of this study were: (i) to determine

whether MeHg and PCB153 alter the MRs in cerebral cortex,

cerebellum, hippocampus and striatum of 21-day-old pups; (ii)

to compare the susceptibility of the offspring brains with that of

the adult brains (dams) to these contaminants; (iii) to assess the

gender-related effects on this endpoint.

2. Materials and methods

2.1. Chemicals

[3H]Quinuclidinyl benzilate ([3H]QNB) and scintillation

fluid were obtained from Perkin-Elmer life Sciences Italy Srl

(Milan, Italy). Several radiolabeled test substance batches have

been used for the assays with specific activity of 42–49 Ci/

mmol and purity >99% by HPLC.

Methylmercury (II) hydroxide (about 1 M aqueous solution,

purity 97%) was purchased from Alfa (Karlsruhe, Germany),

and all other chemicals were from Sigma–Aldrich (Milan,

Italy).

PCB153 was kindly provided by Dr. Ake Bergman

(Department of Environmental Chemistry, Stockholm Uni-

versity). The PCB153 was synthesised by the Ullman diaryl

coupling method, chromatographically isolated and finally

the polychlorodibenzofurans, formed in the reaction, were

removed on a charcoal column (Bergman et al., 1990).

2.2. Animals and treatments

All experimental procedures involving animals were

performed in compliance with the European Council Directive

86/609/EEC on the care and use of laboratory animals.

Sprague–Dawley rats (24 females and 8 males, 12 weeks old

for each set of experiment) were purchased from Charles River

Italia (Calco, Italy) and allowed to acclimatize for 3 weeks.

Throughout the experiment, animals were kept in an artificial

12-h light:12-h dark cycle with humidity at 50 � 10%. Animals

were provided rat chow (4RF21) and tap water ad libitum.

After acclimatization period, three females were placed in a

cage with one male for 24 h. The day after mating was designed

T. Coccini et al. / NeuroToxicology 27 (2006) 468–477470

day 0 of gestation (GD0) and females were housed individually.

Body weight of the dams was measured throughout gestation.

On GD7 the pregnant rats were randomly divided in four

treatment groups (MeHg, PCB153, MeHg + PCB153, control)

as indicated below:

MeHg treatment: MeHg (0.5 or 1.0 mg/kg/day) was

administered to rats (n = 10) in the drinking water, from

GD7 to PND7.

Part of these animals (n = 5) was also exposed to PCB153 as

indicated below.

PCB treatment: PCB153, dissolved in corn oil at the

concentration of 20 mg/ml, was given by gavage, 20 mg/kg/

day, from GD10 to GD16.

Control animals were dosed with the vehicle only (corn oil).

During the treatments, body weight, food and water

consumption were monitored daily. Both adult and immature

animals were sacrificed under CO2 anesthesia on PND21.

Tissues samples: Forebrain areas (cerebral cortex, hippo-

campus, striatum) and cerebellum, were isolated from dams

and offspring and stored at �80 8C until the analyses were

performed. Peritoneal fat (1 g) was also excised for PCB153

analysis.

2.3. MR binding in selected brain areas membranes

MRs were determined by saturation binding assays using the

specific muscarinic antagonist [3H]QNB (Coccini et al., 2000).

Offspring and dams brain areas were pooled from two to four

rats for membrane preparation. The membranes were prepared

by homogenization of the dissected brain area with polytron for

20 s in 20 volumes ice-cold 50 mM Na/K phosphate buffer (pH

7.4) and subsequent centrifugation at 49,000 � g for 10 min at

4 8C. After three washes the resulting membrane pellet was

frozen at �80 8C and resuspended in fresh buffer just prior to

binding assay.

One hundred microlitre atropine (10�5 M) was added

10 min before [3H]QNB to half of the tubes for estimation of

non-specific binding. Brain area membranes (100 ml) were

incubated with [3H]QNB (100 ml) concentrations ranging

from 0.025 to 2 nM in 0.05 M Na/K phosphate buffer (pH 7.4)

in a 2 ml total volume using 96 well plates (a 2-ml capacity/

well; Masterblock, PBI International). Following 60 min

incubation at 27 8C, the reaction was stopped by adding ice-

cold phosphate-buffered saline (PBS). Samples were rapidly

filtered through Unifilter GF/C 96 well plates using a Unifilter

cell harvester (Packard) and washed three times with ice-cold

PBS. Then Unifilter well plates were air dried and counted

for radioactivity in 50 ml of Microscint (Packard) in a Top

Count NXT (Packard) scintillation counter. Each sample was

assayed in duplicate and data were expressed as femtomoles/

milligram of protein.

2.4. Internal quality control

A pooled rat brain preparation obtained from control,

untreated animals, was taken as the internal quality control of

the MR binding assay. This reference value was always tested

in all analytical sessions. The intra-assay coefficient of

variation (CV) was <10% for MR binding in rat brain

calculated on three to six replicates of different aliquots from

the same rat brain membrane pool. The inter-assay CV ranged

between 10 and 15%.

2.5. Analytical measurement of Hg and PCB153 in the

animal tissues

The procedure for mercury analysis in brain tissues has been

previously described (Pineau et al., 1990), but changes in

equipment necessitated some adjustments. The specimen was

freeze-dried for 48 h before determination of the dry weight.

The heating program for the microwave oven was 10 min at

100% power followed by 5 min at 5% and 10 min at 100%

power. The volume of the digested sample used for analysis was

500 ml. The mercury analysis was performed by flow-injection

cold-vapor atomic absorption spectrometry (FIMS-400 and

AS-90 from Perkin-Elmer, Wellesley, MA, USA) (Grandjean

et al., 2005). The standard curve (0, 2, 4 and 6 ug/l) was

generated by dilution of stock standard with 4.3 M HNO3

(containing 5 ml gold solution 1 g/l per liter of HNO3). For

quality assurance of the tissue analyses, reference materials

with low mercury concentrations were analyzed: BCR 184

(bovine muscle) and BCR 185 (bovine liver) (IRMM, Geel,

Belgium). The total analytical imprecision was estimated to be

28% and 8.9% at mercury concentrations of 0.0045 and

0.0392 mg/g, respectively. Given the very low concentrations,

the accuracy was deemed acceptable, with mercury results of

0.0045 mg/g (certified value 0.0026 mg/g) and 0.039 mg/g

(certified value 0.044 mg/g), respectively.

PCB analysis was performed by dual column gas chromato-

graphy (GC3800, Varian; DB-5 and DB-1701 capillary columns,

J & W) with electron capture detectors. Gas chromatographic

operating conditions and samples preparations were similar to

those described previously (Hany et al., 1999). A certified pork

fat (IRMM-446, certified value for PCB153 0,030 mg/kg) was

used for quality control. Successful participation in external

round robins (German EQAS 10 A, 10 B) was certified. PCB

detection limits in brain and adipose samples lay within 0.005

and 0.01 mg/g of extractable fat. The precision was >90% in all

cases. The results were expressed in mg/kg fat.

2.6. Data analysis

Receptor density (Bmax, expressed as femtomoles/milligram

of protein), affinity (defined as the reciprocal of the dissociation

constant, Kd), and the Hill coefficients (nH) were estimated by

nonlinear regression analysis of saturation binding data

according to the method described by Bylund and Yamamura

(1990). Statistical significance was determined by analysis of

variance (ANOVA) and Fisher’s test using SPSS statistical

software. Statistical comparison of Hg or PCB153 concentra-

tions between groups (Hg versus Hg + PCB153 and PCB153

versus PCB153 + Hg) in brain and fat of dams and both

offspring gender was performed using Student’s t test.

T. Coccini et al. / NeuroToxicology 27 (2006) 468–477 471

3. Results

3.1. Developmental data

There was no evidence of overt toxicity in the dams and

pups from any of the treated groups. During gestation and

lactation, body weights of the mothers exposed to PCB153,

MeHg (either at 0.5 and 1 mg/kg), and PCB153 + MeHg did

not differ from those of the controls (data not shown). Birth

number and post-natal growth were not affected by PCB153

and/or any dose of MeHg used. The average number of pups

was similar in all groups: 14 pups in the control group (9 males

and 5 females); 14 pups in the MeHg (1 mg/kg/day) group (9

males and 5 females); 15 pups in the PCB153 group (9 males

and 6 females); 14 pups in the MeHg (1 mg/kg/day) + PCB153

group (7 males and 7 females).

3.2. MR density (Bmax) in selected brain areas

All determinations were done at the end of the lactational

period (PND21), a time at which the total MR binding sites

reach adult levels in rats (Aubert et al., 1996).

3.2.1. Cerebral cortex

The effects of the treatment with 1 mg MeHg/kg/day and

20 mg PCB153/kg/day are shown in Table 1. The common

finding to all treated animals with MeHg was the increased MR

density: 60% in mothers, 27% in male offspring and 50% in

female offspring. MR density in cerebral cortex was also

increased by PCB153 exposure (47% in mothers, 39% in male

offspring and 48% in female offspring). PCB153 + MeHg co-

exposure was similarly associated with an increase in cortical

Bmax (Table 1; Fig. 1). These measured values were 45% in

mothers, 27% in male offspring and 47% in female offspring,

indicating that MeHg and PCB153, when given together, did

not exert any additive or synergistic effects on such receptors

(Table 1; Fig. 1).

No effects on cortical MR density were observed with the

lower MeHg dose (0.5 mg/kg/day from GD7 to PND7) both in

Table 1

Muscarinic receptor density in the cerebral cortex of rats treated with MeHg 1 mg

Control MeHg

(A)

Dams 583.2 � 13.4 931.3 � 86.3*

Offspring

Male 503.2 � 31.2 640.1 � 46.2*

Female 544.7 � 9.9 816.2 � 104.8*

(B)

Dams 503.1 � 72.7 540.2 � 50.0

Offspring

Male 548.6 � 50.5 601.3 � 55.3

Female 513.8 � 50.0 502.6 � 50.0

MeHg was given from GD7 to PND7 and PCB153 from GD10 to GD16. Values are th

rats were pooled. Each offspring group is a pool of tissues from pups of different

milligram of protein.* p < 0.05 statistically different from control.

the mothers and offspring (Table 1). Exposure to PCB153,

20 mg/kg/day, in combination with 0.5 mg MeHg/kg/day still

caused a significant Bmax increase in all treated animals

(Table 1). Bmax values were increased by 42% in dams, and 19%

and 41% in male and female offspring, respectively.

3.2.2. Cerebellum

Treatment with the higher dose of MeHg (1 mg/kg/day)

caused 87% increase in cerebellar MR density in dams

( p < 0.05) (Table 2). Bmax values were also increased by MeHg

in the offspring cerebellum but this occurred in males (27%)

only. The response to PCB153 was different from that of MeHg

in that PCB153 significantly decreased cerebellar MR density

in mothers (27%) as well as in female offspring (31%). In the

experiments with the combined exposure to MeHg (1 mg/kg/

day) and PCB153, the changes in MR density were similar to

those caused by PCB153 alone (Table 2; Fig. 2).

The lower dose of MeHg (0.5 mg/kg/day) did not cause any

change in MR density both in the mother and offspring

cerebella (Table 2). PCB153 (20 mg/kg/day) given in com-

bination with 0.5 mg MeHg/kg/day produced an MR density

decrease similar to that observed when PCB153 was given with

the higher MeHg dose (Table 2).

3.2.3. Other brain areas

In control rats, the hippocampal Bmax values were in the

range of 800–900, 600–700, 550–700 fmol/mg protein for

mothers, male and female offspring, respectively. The striatal

Bmax values of the mothers, male and female offspring ranged

from 800 to 900, 700 to 800, 650 to 750 fmol/mg protein,

respectively. In these brain areas, no changes in MR density

were observed following administration of MeHg, PCB153 and

their combination in adults and in immature animals.

3.3. MR affinity parameters (Kd, nH)

In all brain areas, MeHg and PCB153, alone and in

combination did not affect the dissociation constant values

(Kd) measured in dams and offspring, thus suggesting that the

/kg/day (A) or 0.5 mg/kg/day (B), and PCB153

PCB153 20 mg/kg/day MeHg + PCB153

859.0 � 73.5* 845.7 � 57.0*

697.7 � 49.9* 638.8 � 24.3*

805.6 � 26.6* 799.5 � 7.3*

664.4 � 20.8* 716.4 � 38.0*

656.4 � 26.0* 654.6 � 4.0*

707.7 � 25.6* 723.3 � 34.6*

e mean of three (A) or two (B) separate assays in which tissues from two to four

mothers. Data (mean � S.E.) indicate the Bmax values express as femtomoles/

T. Coccini et al. / NeuroToxicology 27 (2006) 468–477472

Fig. 1. (A–C) Saturation curves of [3H]QNB binding in the cerebral cortex of animals treated with MeHg (1 mg/kg/day, GD7–PND7) and PCB153 (20 mg/kg/day,

GD10–GD16) alone and in combination. Values are the means of duplicate determinations from a representative experiment. Each group is a pool of two to four rats,

and each offspring group is obtained with pups from different mothers. (D) Cerebral cortex percent Bmax changes in dams, male and female offspring. Values are the

mean of three separate assays on tissue samples from rats exposed to MeHg (1 mg/kg/day, GD7–PND7) and PCB153 (20 mg/kg/day, GD10–GD16) alone and in

combination. *p < 0.05 statistically different from control.

affinity of the ligand [3H]QNB for its receptor was not modified

by these compounds. Control Kd values were for cerebral cortex

0.28 � 0.02, 0.24 � 0.01, 0.48 � 0.09 nM in dams, male and

female offspring, respectively; for cerebellum 0.085 � 0.02,

0.078 � 0.01, 0.080 � 0.01 nM in dams, male and female

offspring, respectively; for hippocampus 0.23 � 0.04, 0.21 �0.03, 0.19 � 0.03 nM in dams, male and female offspring,

Table 2

Muscarinic receptor density in the cerebellum of rats treated with MeHg 1 mg/kg

Control MeHg

(A)

Dams 82.5 � 17.2 154.0 � 3.2*,#,§

Offspring

Male 119.1 � 23.7 151.7 � 4.7*

Female 126.9 � 14.0 115.9 � 7.2

(B)

Dams 80.9 � 8.0 75.0 � 8.6

Offspring

Male 99.9 � 3.9 113.6 � 19.3

Female 123.9 � 9.2 111.9 � 15.9

MeHg was given from GD7 to PND7 and PCB153 from GD10 to GD16. Values are th

rats were pooled. Each offspring group is a pool of tissues from pups of different

milligram of protein.* p < 0.05 statistically different from control.# p < 0.05 statistically different from PCB153.§ p < 0.05 statistically different from MeHg + PCB153.

respectively; for striatum 0.17 � 0.02, 0.21 � 0.03, 0.21 �0.03 nM in dams, male and female offspring, respectively.

The Hill coefficient (nH) values in were close to unit in all

groups indicating homogeneous affinity of the ligand [3H]QNB

for the receptors. In particular, control nH values for dams’ and

pups’ cerebral cortex and hippocampus were 0.99 � 0.01, for

dams’ and pups’ striatum were 0.96 � 0.03, for cerebellum

/day (A) or 0.5 mg/kg/day (B), and PCB153

PCB153 20 mg/kg/day MeHg + PCB153

60.3 � 10.7* 59.4 � 10.8*

106.4 � 26.2 99.0 � 10.9

88.1 � 2.8* 109.1 � 13.4

62.2 � 3.7* 63.7 � 13.5*

110.5 � 7.0 108.3 � 9.0

97.3 � 6.0* 103.6 � 15.7

e mean of three (A) or two (B) separate assays in which tissues from two to four

mothers. Data (mean � S.E.) indicate the Bmax values express as femtomoles/

T. Coccini et al. / NeuroToxicology 27 (2006) 468–477 473

Fig. 2. (A, B and C) Saturation curves of [3H]QNB binding in the cerebral cortex of animals treated with MeHg (1 mg/kg/day, GD7–PND7) and PCB153 (20 mg/kg/

day, GD10–GD16) alone and in combination. (D) Cerebellum percent Bmax changes in dams, male and female offspring. Values are the mean of three separate assays

on tissue samples from rats exposed to MeHg (1 mg/kg/day, GD7–PND7) and PCB153 (20 mg/kg/day, GD10–GD16) alone and in combination. *p < 0.05

statistically different from control. #p < 0.05 statistically different from PCB153. §p < 0.05 statistically different from MeHg + PCB153. Values are the means of

duplicate determinations from a representative experiment. Each group is a pool of two to four rats, and each offspring group is obtained with pups from different

mothers.

Table 3

Total mercury levels in brain of animals treated with MeHg 1 mg/kg/day (GD7–

PND7), PCB153 20 mg/kg/day (GD10–GD16), alone and in combination

Group Brain (mg/g)

Dams Offspring

Male Female

Control 0.013 0.002 0.003

PCB153 0.026 0.024 (0.039), n = 3 0.014 (0.024), n = 6

MeHg 7.53 1.67 (0.43), n = 4 1.52 (0.33), n = 4

MeHg + PCB153 8.78 2.80 (1.81), n = 6 1.63 (0.20), n = 6

Values are the mean � S.D. (in parenthesis).

Table 4

PCB153 levels in peritoneal fat and brain of animals treated with MeHg 1 mg/kg/day

Group Fat (mg/kg)

Dams Offspring

Male Female

Control 0.009 0.093 (0.062), n = 8 0.101 (

PCB153 291.60 (89.90), n = 5 341.63 (126.44), n = 16 288.12

MeHg 0.072 (0.04), n = 2 0.139 (1.108), n = 7 0.101 (

MeHg + PCB153 324.75 (31.02), n = 4 482.22 (267.17), n = 18 422.58

Values are the mean � S.D. (in parenthesis). NA, not available.* p < 0.05 statistically different from PCB153 group.

0.95 � 0.02, 0.94 � 0.05, 0.96 � 0.03 nM in dams, male and

female offspring.

3.4. Hg and PCB153 retention

Hg levels were measured in whole brains of both dams and

pups (separate pools accordingly to gender) treated with

1 mg MeHg/kg/day. The Hg concentrations (Table 3) in the

brain were about five-fold higher in the mother than in the

offspring. The Hg retention was not affected by the co-exposure

to PCB153.

PCB153 levels were measured in fat and whole brain of both

dams and pups.

(GD7–PND7), PCB153 20 mg/kg/day (GD10–GD16), alone and in combination

Brain (mg/kg)

Dams Offspring

Male Female

0.064), n = 11 0.096 0.241 (0.234), n = 2 0.090 (0.077), n = 3

(89.76), n = 17 2.87 25.16 (6.23), n = 3 31.96 (19.47), n = 5

0.044), n = 6 0.021 0.136 (0.070), n = 5 0.083 (0.036), n = 5

(212.96)*, n = 19 NA 66.15 (29.07)*, n = 6 49.68 (16.30), n = 6

T. Coccini et al. / NeuroToxicology 27 (2006) 468–477474

The levels of PCB153 (Table 4) in the fat were similar in the

mothers and their pups, while this compound was much more

concentrated (8–10 times) in the brains of pups than in their

mothers. MeHg co-exposure seemed to increase both fat and

brain PCB153 concentrations in pups as compared to the group

exposed to PCB153 only.

4. Discussion

This study demonstrates that at weaning rat brain MRs

were modified following developmental exposure to MeHg

at 1 mg/kg bw/day from GD7 to PND7 and/or PCB153 at

20 mg/kg bw/day from GD10 to GD16. The cerebral cortex

and cerebellum appeared to be the most sensitive areas to

both compounds. Notably, the MRs changes were detected

also in adult animals 2 weeks after cessation of MeHg treat-

ment. Moreover, gender-related differences in the offspring

were found in the cerebellum after both MeHg and PCB153

exposure.

When a lower dose of MeHg (0.5 mg/kg/day) was

administered using the same timing of perinatal treatment

protocol, no effect on brain MRs was observed in the immature

and mature animals.

4.1. MeHg effects

The major common finding following MeHg exposure at the

dose of 1 mg/kg/day, from GD7 to PND7, was an increased MR

density at PND21 in two target brain areas, namely cerebral

cortex and cerebellum of both mothers and offspring, except for

the female pups’ cerebellum showing no changes in MR density

as compared to controls. Our previous MR studies in female

adult rats showed similar dose-dependent increases in brain

MRs 2 weeks after the last MeHg treatment (0.5–2 mg/kg/day

for 14 days) (Coccini et al., 2000). The delayed up-regulation of

the cerebral MRs observed here after repeated MeHg exposure

may reflect compensatory mechanism for early-stage choli-

nergic effects. The latter may include (i) the inhibition of

acetylcholine (ACh) synthesis by MeHg and consequent

reduction of brain ACh levels and (ii) a direct competitive

antagonism of MeHg at MRs. MeHg has been found to inhibit

choline acetyltransferase (ChAT) activity as well as ACh

synthesis in target rodent brain areas such as cerebellum and

cerebral cortex (Hrdina et al., 1976; Kobayashi et al., 1979;

Tsuzuki, 1981). In addition, several in vitro studies have

indicated that MeHg can directly affect MR binding (Abd-

Elfattah and Shamoo, 1981; Basu et al., 2005; Castoldi et al.,

1996; Eldefrawi et al., 1977; Von Burg et al., 1980). In in vitro

experiments using cortical membranes, we have previously

demonstrated a direct MeHg interaction with the M1 and M2

receptor subtypes. The effect of MeHg was observed at

concentrations ranging from 3 to 150 mM (Castoldi et al.,

1996), which are comparable to the levels reached in rat brain

(mothers and offspring) after the in vivo doses used in the

present study (8–50 mM calculated from data of Table 3). The

recent hypothesis that degeneration of cerebellar granule cells

by MeHg is mediated by activation of the M3 MR subtype

(Limke et al., 2004) further stresses the relevance of MRs in

MeHg-induced neurotoxicity.

MR density in all brain areas of control weaning male and

female pups was similar to that determined in the respective

brain areas of their mothers (Tables 1 and 2) according to the

ontogenesis of these receptors in the rat forebrain (Aubert et al.,

1996).

A more pronounced increase in MR density by MeHg was

observed in the mothers’ brain (87% and 60% in cerebellum

and cerebral cortex, respectively) than in male littermates (27%

in both areas). Notably, at PND21 whole brain mercury levels

were markedly different between rat dams (7–9 mg/g) and pups

(1.5–2.8 mg/g), thus suggesting that toxicokinetic factors may,

at least in part, underlie the stronger MeHg effect on MRs in the

adult brain. Literature data have shown that, under continuous

gestational and lactational exposure to 5 ppm MeHg in diet, the

metal concentration in the offspring brain rapidly declines post-

natally from 4.5 mg/g at birth to 1 mg/g at PND20 (suggesting a

limited MeHg absorption from maternal milk) at variance with

the constant levels detected in the lactating mother’s brains

from day 0 to day 21 post-partum (4 and 3.5 mg/g, respectively)

(Sakamoto et al., 2002).

Other mechanisms, such as gender-related differences in

MR susceptibility may explain the different response to MeHg

observed in our study, considering that brain Hg levels were

comparable in male and female pups, differently from the

extent of MR Bmax changes detected in their cerebral cortex as

well as cerebellum. In the study by Gimenez-Llort et al. (2001)

changes in dopamine-modulated motor activity were observed

in male, but not female rats despite the presence of similar brain

MeHg concentrations in both genders.

4.2. PCB153 effects

The administration of PCB153 alone during pregnancy

increased and decreased MR density in cerebral cortex and

cerebellum, respectively, in both mature and immature animals.

To explain such opposite regional effects, the following

hypotheses might be considered: (i) intrinsically, distinct MR

subtypes may not be equally susceptible to PCB153; thus, the

regional effects of PCB153 may reflect differences in the

expression of MR subtypes between the cerebral cortex and the

cerebellum, with the first one expressing mostly M1 and M3

receptors, and the second one being almost exclusively

composed by the M2 subtype with the M3 expressed only in

cerebellar granule cells (Volpicelli and Levey, 2004; Limke

et al., 2004); (ii) in addition, the interaction of PCB153 with

post-synaptic M1 and pre-synaptic M2 subtypes may differ-

ently modulate ACh release and levels leading to distinct

compensatory up- or down-regulation of MR density; (iii) the

different stages of maturation reached by MR subtypes at the

time of PCB exposure may also play a role in the PCB-area

dependent effects: in this respect, M2 receptors develop after

the M1 subtype, the latter reaching the adult values between

PND21 and PND35, while M2 after PND35 (Aubert et al.,

1996); (iv) based on the effects of PCB on the dopaminergic

system (Chu et al., 1996), the altered MRs might result

T. Coccini et al. / NeuroToxicology 27 (2006) 468–477 475

indirectly from the interactions between the dopaminergic and

muscarinic cholinergic systems.

Behavioural alterations, reduced learning ability and

reduced long-term potentiation have been described in rats

exposed to PCB153 during development (Schantz et al., 1995;

Holene et al., 1998; Hussain et al., 2000). The possibility exists

that changes in MRs contribute to the onset of PCB153-induced

neurodevelopmental deficits.

To our knowledge, the effect of the individual PCB153

congener on the muscarinic system has not been investigated

before. Documented changes on this neurotransmission system

include depression of rat cerebral ChAT activity following

developmental administration of Aroclor 1254 (Juarez de Ku

et al., 1994), and changes in murine brain MR density by

PCB77 (Eriksson, 1988; Eriksson et al., 1991).

PCB153 concentrations, in the exposed dams, were about

3 mg/kg fat in the brain and averaged about 300 mg/kg fat in the

adipose tissue at day 21 post-partum (Table 4). This distribution

between the brain and the fat compartments well compares with

literature data showing in adult animals fat/brain PCB content

ratios in the range of 50–110 following prolonged exposure

(Chu et al., 1996; Kaya et al., 2002).

On PND21 the levels of PCB153 in fat were similar in the

mothers and their pups, while in brain PCB values were 8–10

times higher in the pups than in their mothers. Notably, Kaya

et al. (2002) demonstrated that from parturition to weaning,

PCB concentrations increase in the brain of suckling rat pups,

while they simultaneously decrease in the brain of their

lactating mothers exposed per os to a PCB mixture (0.5, 2 or

4 mg/kg bw/day) from day 50 before mating until delivery.

These data indicate that lactation is a relevant route of exposure

to PCB for the immature organism. Another recent study has

confirmed that the absolute quantity of PCB153 transferred via

breast milk is higher than the quantities transferred via placenta

(Lee et al., 2002).

The quantitative comparison, between adults and rat

pups, of MR changes caused by PCB apparently indicates a

similar degree of effects despite the higher levels of

contaminant detected in the whole brain of young animals.

Notably, a single administration of 0.41 or 41 mg/kg PCB77

at PND10 caused an early change in mouse brain MR density

which was not dose-related, despite the 100-fold difference

between the two selected doses (Eriksson, 1988). In mice

exposed to the higher dose, the altered MR density (+5%) was

still determined at the age of 4 months and this effect was

accompanied by changes in spontaneous motor behaviour

(Eriksson et al., 1991).

4.3. MeHg + PCB153 effects

To our knowledge, the present study provides the first in vivo

documentation that PCB153 can change but does not

exacerbate the central effects of MeHg after developmental

co-exposure. Previous in vivo and in vitro investigations have

instead indicated possible additive effects of PCB mixtures

(Aroclors) and MeHg (Roegge et al., 2004; Bemis and Seegal,

1999).

According to our data, the MeHg (1 mg/kg/day) + PCB153

co-treatment (a) induced an increase in the receptor number

similar in extent to that caused by either compound given alone

in the cerebral cortex of all age/gender groups; (b) completely

masked MeHg-induced increase in MR number in the

cerebellum of dams and male offspring.

Altogether, these results indicate that non-additive interac-

tions occurred between MeHg and PCB153 regarding the

effects on MRs in both the cerebral cortex and cerebellum. The

combined effect of MeHg and PCB153 cannot be ascribed to

pharmacokinetic mechanisms, as suggested by the results of

analytical studies. Indeed, brain Hg levels were comparable in

the groups of MeHg alone and MeHg + PCB153. Relatively to

PCB153, the fat and brain levels were higher in the co-exposed

pups than in those treated with PCB153 alone although the

extent of MR changes was similar in the two groups.

MeHg and PCBs have been previously hypothesized to

participate in the same mechanism of toxicity, involving the

ryanodine receptor, causing either synergistic or antagonistic

effects on intracellular calcium levels depending on the

compound concentrations and duration of cell exposure (Bemis

and Seegal, 2000). In our study, PCB153 was given in a higher

amount and in a narrower time of window compared to MeHg.

On a molar basis, in the brain of co-exposed pups MeHg and

PCB153 concentrations were about 10 and 140–180 mM,

respectively. Supposing that both contaminants act on the same

molecular target, the 14- to 18-fold higher PCB153 concentra-

tion compared to that of MeHg may explain why the first agent

masks the effect of organic mercury on MRs. Alternatively, if

they act on different molecular targets, the high levels of

PCB153 may cause conformational changes in MeHg binding

sites that prevent it from exerting its effect, as previously

hypothesized by Bemis and Seegal (2000). Still, PCBs and

MeHg may affect a range of other functions in the brain, and the

extent of overlapping mechanisms and potentials for synergism

are by no means clear (Costa et al., 2004). For example, there

are reports indicating that thyroid hormone imbalance and cell

signalling alterations play a role in the neurotoxic effects of

PCBs and MeHg (Castoldi et al., 2001; Roelens et al., 2005;

Tilson and Kodavanti, 1997). It has been proposed that PCBs

can affect brain development by interfering with thyroid

hormone (TH) signalling as well as by interacting with the

cerebral expression of TH-regulated genes (Roelens et al.,

2005). Interestingly, in experimental models the alteration of

thyroid state has been found to affect the maturation of a

number of neurochemical endpoints including acetylcholines-

terase activity and [3H]QNB muscarinic binding (Virgili et al.,

1991). PKC translocation is recognized as a sensitive target for

the ortho-substituted PCBs (Tilson and Kodavanti, 1997) and

PKC activity appears to be targeted by MeHg as well according

to in vitro evidence (Parran et al., 2004).

In the present study, the dose of 1 mg MeHg/kg/day

markedly affected the rat cholinergic system. After such

treatment, cerebral Hg levels in weaning rats were 1.5–2.8 ppm

(Table 3) that are 5–10 times higher than Hg levels detected in

the brain of environmentally exposed human newborns (from

Lewandowski et al., 2003).

T. Coccini et al. / NeuroToxicology 27 (2006) 468–477476

The study also shows that PCB153 alters, in the developing

brain, central cholinergic (muscarinic) system which is known

to play a role in many fundamental CNS functions. Noteworthy,

the total dose given to rat dams (140 mg/kg) was similar to the

total maternal PCB153 dose (175 mg/kg) that caused sig-

nificant reduction of LTP in the rat offspring (Hussain et al.,

2000) and resulted in maternal serum levels of 1125 � 490 ppb.

For comparison with environmental human exposure, this level

is about 10-fold higher than the median PCB153 human serum

levels (110 ng/g serum lipid) detected among 10 studies of

PCBs and neurodevelopment, and is 2.5-fold higher than those

determined in Faroese population children (450 ng/g) (Long-

necker et al., 2003).

Acknowledgements

The present study was supported by the European

Commission (Quality of Life and Management of Living

Resources Programme QLK4-CT-2001-00186 and FOOD-CT-

2003-506543) and the Italian Ministry of Health. Dr. Aake

Bergman (Stockholm University) kindly provided PCB153

synthesized in his laboratory. The authors wish to thank Mr.

Davide Acerbi for his excellent technical assistance.

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