FUNDAMENTAL AND APPLIED TOXICOLOGY 15, 457 -467 ( 1990)

Behavioral Effects of Pre- and Postnatal Exposureto a Mixture of Low Chlorinated PCBs in Rats

HELLMUTH LILIENTHAL, MANFRED NEUF, CARMEN MUNOZ,1 AND GERHARD WINNEKE

Medical Institute of Environmental Hygiene at the University ofDusseldorf, A ufm Hennekamp 50,D-4000 Dusseldorf1, Federal Republic of Germany

Received December 15, 1989; accepted May 9, 1990

Behavioral Effects of Pre- and Postnatal Exposure to a Mixture of Low Chlorinated PCBs inRats. LILIENTHAL, H., NEUF, M., MUNOZ, C, AND WINNEKE, G. (1990). Fundam. Appl. Toxi-col. 15, 457^67. Polychlorinated biphenyl (PCB)-treated Wistar rats were tested on threedifferent behavioral paradigms. Animals were pre- and postnatally exposed to a technical mix-ture of PCBs with a chlorine content of 42%. Exposure levels were 0, 5, or 30 mg/kg diet. Theseconditions did not affect the health of the dams, the litter size or weight, or the physical develop-ment of the offspring. Relative liver weights in the offspring, however, were elevated in a dose-dependent manner. Open-field ambulation, active avoidance learning, and operant condition-ing on a fixed interval 30-sec schedule (FI-30-sec) were used to evaluate PCB-induced behavioralalterations. Ambulation was increased in 30-mg-treated rats at Day 22, but not at Day 120.There were more avoidance responses and intertrial responses in the 30-mg group than in bothother groups. On the FI-30-sec schedule slightly more reactions were emitted by the 30-mg groupduring the first 10 sec of the interval than by the other animals. More pronounced, however, werethe differences between groups in the temporal pattern of responses within the 30-sec interval. Itis concluded that in rats PCB exposure causes consistent alterations in all of the tested activity-dependent behaviors. C 1990 Society of Toxicology.

Although production of polychlorinated bi- human neonates is limited. Some publicphenyls (PCBs) has stopped in most coun- health studies suggest that gestational age andtries, these substances persist in the environ- neuropsychological development are affectedment due to their chemical and thermal sta- (Fein et al., 1984; Rogan et al, 1986b), andbility and longevity. They accumulate in food the nervous system has been pointed out aschains and are found in considerable one of the target organs. Babies previously ex-amounts in human milk (e.g., DFG, 1988; P 0 ^ t 0 PCBs a n d t e s t e d a t t h e a 8 e of aboutRogan et al., 1986a, Weisenberg et al., 1985; 7 months perform less well on visual recogni-Yakushiji et al., 1979) in industrialized coun- t i o n memory testing (Jacobson et al, 1985)tries. The safety factors are small and the con- w n i c n ^"^^ f o r cognitive development andcentration level in human milk has remained <*n ^ r ega l ed as a valid predictor of laterstable during recent years in Western Europe intelligence (Pagan and McGrath, 1981). At(DFG, 1988). Until now, our knowledge of m u c h h i e h e r exposure levels people who hadthe effects of PCB exposure at low levels in s u f f e r e d f r o m rcB poisoning by ingesting

contaminated rice oil exhibited slowed motorand sensory nerve conduction velocities even

' PresentaddressMnstituteofToxicologyandEmbryo- years after the accident (Chia and Chu, 1985).nal Pharmacology, Garystrasse 5, D-1000 Berlin 33, 1° experimental studies in mice it has beenFRG. shown that PCBs cause hyperactivity, stereo-

4 5 7 0272-0590/90 $3.00CopyrifbtO 1990 by the Society of Toxicology.All rights of reproduction in any form reservtd.

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458 LILIENTHAL ET AL.

typic circling, and decreased motor coordina-tion (Chou et al, 1979; Tilson et al., 1979;Storm et al., 1981). In monkeys PCB-in-duced early developmental hyperactivitychanged to hypoactivity with aging (Bowmanand Heironimus, 1981). Discriminationlearning was also impaired in monkeys (Bow-man et al., 1978). As there is only limited in-formation about the neurobehavioral effectsof long-term low-level exposure to PCBs andbecause laboratory studies of such effectscould have relevance for human babies, thepresent investigation was started using pre-and postnatal exposure in rats. Despite thefrequent use of the rat in studies of enzymeinduction by PCBs (for a review see Safe,1984), only a few studies of behavioral effectsare reported in this species (Shiota, 1976; Ov-ermann et al., 1987, Pantaleoni et al., 1988).Three different experimental paradigms,each known to be dependent on the activitylevel, were used. These were activity in theopen field, active avoidance learning in theshuttle box, and operant conditioning on afixed interval schedule.

METHODS

Animals and Treatments

Twenty-four female Wistar albino rats weighing 200-220 g (Ivanovas, KJeslegg, FRG) were housed in plasticcages (four rats per cage). A 12-hr light cycle from 3 AMto 3 PM was maintained, room temperature was 23"C,and the relative humidity was 55%. The rats were ran-domly assigned to three groups of eight subjects and ex-posed to 0, 5, or 30 mg PCB/kg diet. The commercialPCB mixture (Clophen A30, Dr. Ehrenstorfer, Augs-burg, FRG) was added to the laboratory diet (Altromin1314) by the manufacturer (Fa. Altrogge, Lage, FRG).The contaminated diets were prepared three times dur-ing the experiments. Due to the thermal and chemicalstability and low volatilization of PCBs there were nospecial requirements for storing the diets. Deviationsfrom the concentrations given above were within the10% range. PCBs were not detected in the control diet(limit 0.1 mg/kg diet). The exposure started 60 days priorto mating for which two females were kept together withone hitherto nonexposed male for 18 days. This treat-ment was terminated for mothers weaning offspring atPostnatal Day 21 (PND 21). The offspring received the

same PCB-containing diets as their mothers after wean-ing. Water was supplied ad libitum throughout the exper-iment for all animals.

The particular mixture of low chlorinated PCBs waschosen and the concentrations were adjusted in order toavoid possible effects on reproduction (Brezner et al.,1984; Keplinger et ai, 1971) as well as gross toxicity.Since the rat, like the mouse and the hamster, toleratesgreater amounts of PCBs than certain other species, in-cluding subhuman primates, treatment levels are highwhen compared to human exposure: The diets with 5 or30 mg PCB/kg result in an intake of about 0.4 or 2.4 mg/kg body wt, respectively, for a rat weighing 250 g andeating a 20 g diet per day. In comparison, the averagedaily intake via food by human adults is about 0.08 ngjkg body wt and an order of magnitude higher in babies(DFG, 1988).

Experimental Apparatus

A white painted octagonal open field was used for theactivity measurements. It was illuminated by an incan-descent lamp (25 W) hanging 1 m above the floor of thedevice. The floor was equally divided into 25 segments,namely, 1 center field and two concentric rings of 8 and16 radial segments for the inner and the outer ring, re-spectively. The diameter of the device was 35 cm for ani-mals tested on PND 22 and 75 cm for rats tested on PND120 (see Behavioral Procedures below).

Active avoidance learning (AAL) was studied in ashuttle box. A description of the apparatus and proce-dures is given elsewhere (Winneke et al., 1982). Briefly,the box was divided into two compartments. It was com-puter-controlled (Rockwell AIM 65) and placed in asound attenuated chamber which reduced external noiseby 40 dB. Scrambled electric footshock was provided bya shock generator and scrambler (Campden Instruments,Ltd., London, UK).

Two standard operant conditioning chambers (Camp-den Instruments) situated in sound attenuated chambersserved for the examination of fixed interval performance.They measured 20.5 X 24.5 X 22.5 cm (H X W X D).Each of them was equipped with two levers of which onlythe left one was used. The force required for operatingthe lever was about 18 mN and a displacement of 3 mmwas necessary for closing the electromagnetic switchwhich caused the delivery of a 45-mg dustfree Bioservfood pellet by a Campden pellet dispenser. Cumulativerecorders were used to monitor the daily performanceof the animals. The whole setup was controlled by twoRockwell AIM 65 microcomputers.

Behavioral Procedures

General. All testing was done with the offspring. Naiverats were used in each of the behavioral tests employed.

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PRE- AND POSTNATAL PCB EXPOSURE 459

They were randomly selected and no more than two ani-mals were taken from a litter for examination on one par-adigm. Only males were used in order to exclude possibleinfluences of the estrous cycle on activity.

Open field. At PND 22 male animals (10 per group)were tested in the smaller open field for 9 min. The ses-sion was recorded on video tape and evaluated by a tech-nician unaware of the exposure condition. Crossed seg-ments were counted and summated across 3 min. Thecriterion for a count was that the rat had crossed a linewith at least three feet. Testing started at 4:00 PM. Thesame procedure was followed at PND 120 using thelarger open field.

Active avoidance learning (AAL) At PND 65-75 malerats (12 per group) were examined for two-way AAL. Ascrambled electric footshock of 0.5 raA was given for amaximum of 15 sec. It was preceded by a 4-kHz tone (75dB SPL). This warning stimulus was given 5 sec beforeshock onset and lasted until the shock was terminated bythe rat running from one compartment to the other. Anavoidance reaction was recorded when the rat changedcompartments during the 5 sec of the warning stimulusbefore shock delivery. An avoidance reaction terminatedboth tone and shock. Ten consecutive avoidance reac-tions were the criterion for successful learning afterwhich the program stopped and the rat was removedfrom the chamber. The intertrial interval was 30 sec.Eighty trials per day were given to each animal on 2 con-secutive days. Responses in the intertrial interval werenot punished, but recorded.

Operanl conditioning. Operant conditioning started atPND 380. Male rats (6 per group) were placed in singlecages and the average daily food intake was determinedover 1 week. The animals were then deprived of food for24 h. Thereafter they were trained for 2 days to press theleft lever to obtain food pellets. Each training session wasterminated after 40 min. The animals were then giventwo sessions in which they worked on a continuous rein-forcement (CRF) schedule. Each of these sessions wasterminated after 30 min or when the rat had received 150pellets. The animals were then switched to a fixed inter-val 30-sec schedule of reinforcement (FI-30-sec) onwhich the first bar press after 30 sec from the previousreinforcement was rewarded by food delivery. In a singlesession each rat was given 90 pellets which resulted in asession duration of 45-50 min. Thereafter the animalwas removed to its home cage and given 80% of its nor-mal daily intake as food supplement. Each animal wasgiven 25 daily sessions.

Staiistical A nalysis

Groups were compared by analysis of variance withrepeated measures (activity and AAL) using the SASpackage. Post-hoc comparisons were made with Tukey'sstudentized range test. Because of the time-consuming

operant conditioning procedure only a small number ofanimals per group could be used in this experiment.Therefore, a test based on random assignment is moreappropriate than a test based on the assumption of ran-dom sampling. For the randomization test the I statisticwas used (Edgington, 1980). For evaluation of the re-sponse pattern of the animals spline functions were used(Stoer and Bulirsch, 1980). The average distribution ofreactions within the 30-sec interval was calculated foreach of the 25 sessions by summation of the reactions ineach 10-sec part of the interval across the 90 trials. Thisprocedure yielded 3 values for each day or 75 values forall sessions. Cubic regular splines were fitted to the result-ing response curves using the SAS/IML routine.

Determination ofPCBs

Before the open field was run at PND 120 and at PND420 after the operant conditioning, three male animalswhich had not taken part in the behavioral experimentswere randomly drawn from each exposure group andkilled by ether inhalation. Brains and perirenal fat wereremoved and deep frozen for PCB determination. Organcontents of PCBs were determined by an official labora-tory for analytical chemistry (Dr. Specht and Partner,Hamburg, FRG). The pattern of Clophen A30 was nolonger detected in the organ probes. Therefore, onlythose six components were determined which are mea-sured as indicators for the PCB level in food according tothe Directive on MRLs in FRG. These were the No. 28,52, 101, 138, 153, and 180(BallschmiterandZell, 1980),namely, 2,4,4'-triCB, 2,2',5,5'-tetraCB, 2,2',4,5,5-pen-taCB, 2,2',3,4,4',5'-hexaCB, 2,2',4,4',5,5'-hexaCB, and2,2',3,4,4',5,5'-heptaCB, respectively. These six conge-ners account for about 60% of the total PCB content inbutter and human milk (Schulte and Malisch, 1984). Or-gan contents were determined using capillary chroma-tography with an ECD detector on two or three columnsof different polarity (Specht and Tillkes 1980, 1985). Thecontamination of the commercial PCB mixture used(Clophen A30) was analyzed by the same laboratory asabove. It revealed a concentration of 2.479 mg totalPCDF/kg PCBs (tetraCDF 1.708 mg/kg, pentaCDF0.572 mg/kg, hexaCDF 0.177 mg/kg, and heptaCDF0.022 mg/kg). This results in a concentration of about 75ng total PCDF/kg in the diet with 30 mg PCB/kg. PCDDswere not detected (detection limit 0.5-2 fig/kg PCBs).

RESULTS

Somatic Development and PCB Contents

All of the eight females per group deliveredwithin 3 weeks after termination of the mat-

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460 LILIENTHAL ET AL.

No.

2852

101138153180Sum

2852

101138153180Sum

2852

101138153180Sum

Brain

n.d.n.d.n.d.n.d.n.d.n.d.—

n.d.n.d.n.d.n.d.n.d.n.d.—

TABLE 1

TISSUE CONCENTRATION OF PCB CONGENERS

Controls

Adipose

0.03 ± 0.06n.d.n.d.n.d.n.d.n.d.0.03

0.06 ± 0.02<0.01<0.01

0.07 ± 0.030.07 ± 0.030.02 ±0.01

0.23

5-mg group

Brain

Age 120 days (n =

0.03 ±0.01n.d.n.d.

0.01 ±0.010.01 ±0.01

n.d.0.05

Age 420 days (n =

0.06 ± 0.020.01 ±0.01

<.010.01 ±0.010.01 ±0.00

<0.010.10

Adipose

3/group)

2 .37 ± 1 . 1 50.06 ± 0.040.04 ±0.010.63 ± 0.040.55 ±0.170.22 ± 0.04

3.87

3/group)

2.66 ±0.720.04 ± 0.030.06 ± 0.000.72 ±0.130.56 ±0.130.27 ±0.06

4.31

30-mg group

Brain

0.07 ± 0.03n.d.n.d.

0.05 ± 0.020.04 ±0.010.02 ±0.01

0.18

0.11 ±0.080.01 ±0.02

<0.010.05 ± 0.020.04 ± 0.020.02 ± 0.02

0.23

Age 420 days, exposure free interval during operant behavioral testing (n = 2/group)

n.d.n.d.n.d.n.d.n.d.n.d.—

0.02 ±0.010.02 ±0.01

<0.010.08 ± 0.030.09 ± 0.030.02 + 0.01

0.23

n.d.n.d.n.d.

0.01 ±0.00<0.01<0.01

0.02

0.02 ± 0.030.01 ±0.010.01 ±0.010.50 ± 0.660.40 ±0.530.23 ±0.32

1.17

Note. Values (means ± SD) in mg PCB/kg tissue; n.d., not detected (limit = 0.01single congeners according to Ballschmiter and Zell (1980): 28, 2,4,4'-triCB;2,2',4,4',5,5'-hexaCB; 52, 2,2',5,5'-tetraCB; 138, 2,2/,3,4,4')5'-hexaCB; 180, 2,2',3,4,4',

n.d.n.d.n.d.

0.02 ±0.010.03 ±0.000.01 ±0.00

0.06

Adipose

5.26 ±0.520.14 ±0.030.11 ±0.014.51 ±0.823.22 ±0.501.78 ± 0.18

15.02

6.58 ±2.810.13 ±0.060.16 ±0.023.89 ±0.343.46 ±0.192.05 ±0.11

16.27

0.02 ± 0.00n.d.

0.01 ±0.012.20 ±1.042.82 ±0.121.38±0.19

6.43

mg/kg). The numbers represent101, 2,2',4,5,5-pentaCB; 153,

,5,5'-heptaCB. See text (determi-nation of PCBs under Methods) for further information.

ing period. There were 68, 83, and 59 pupsborn in the control, 5-mg, and 30-mg groups,respectively. The treated groups did not differsignificantly in litter size in comparison tocontrols (Tukey, p > 0.1). Therefore, we de-cided not to standardize the numbers per lit-ter. There were no PCB-dependent decreasesof body weight at any time in the experiment.The PCB contents in brains and perirenal fatare shown in Table 1. There was a dose-re-lated increase in the PCB level of all conge-ners in both brain and fat tissues. The content

of PCBs was much higher in the adipose thanthat in the brain tissue. This table also dem-onstrates the higher accumulation of hexa-and heptachlorinated congeners since theseare found at levels <1% in the technical mix-ture used (Schulte and Maliscb, 1983). At theend of the exposure free interval during oper-ant behavioral testing low chlorinated conge-ners are remarkably reduced even in the 30-mg group. The liver to body weight ratio wasincreased in a dose-dependent manner in ex-posed animals (Table 2).

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PRE- AND POSTNATAL PCB EXPOSURE 461

TABLE 2

LIVER TO BODY WEIGHT RATIO

IN OFFSPRING ANIMALS

Age 120 days Age 420 days

Controls5-mg group30-mg group

0.0407 ± 0.00680.0436 ± 0.00030.0509 ± 0.0072

0.0401 ±0.00300.0425 ±0.00150.0483 ± 0.0027

Note, n = 3; means ± SD.

Open-Field Activity

The results for activity in the open field atPND 22 are shown in Fig. 1 (left). Analysis ofvariance with repeated measures on the timefactor revealed significant overall differ-ences between the groups (F\2,27) = 6.96; p< 0.005). Activity levels were significantlyraised by exposure to 30 mg/kg in compari-son to those with both of the other treatmentconditions (Tukey's studentized range test, p< 0.05). The interaction between the PCBand the time factor was not significant, indi-cating that habituation was not changed bythe treatment. At PND 120 activity levels in

the open field were almost identical under alltreatment conditions (Fig. 1, right).

Active A voidance Learning

Rats treated with a 30 mg PCB/kg diet ex-hibited an improved AAL performance ascompared to controls and the 5-mg group(Fig. 2). There was a significant overall PCBeffect according to analysis of variance withrepeated measures on the trial factor only forthe first day of learning CF(2,33) = 8.26; p< 0.005). The interaction between the treat-ment and the trial factor was significant aswell (fl(14,231) = 2.13;/? < 0.05). This is dueto the different shapes of the learning curvesafter PCB exposure. Post-hoc analysis re-vealed significant differences between the 30-mg group and both of the other groups (Tu-key's studentized range test, p < 0.05). Thetrials-to-criterion measure failed to reach sig-nificance according to analysis of variance(^2,33) = 2.16; p > 0.1). However, relativeintertrial crossings, i.e., intertrial crossings di-vided by trials-to-criterion, were significantly

100

' 8 0

60

40

20

100

80-

60

40

2 0 '

9min9min

FIG. 1. Open-field activity at the ages of 22 (left) and 120 days (right); (circles) controls; (triangles) 5-mggroup; (squares) 30-mg group. For evaluating ambulation the number of crossed segments was counted(means ± 95% CL). The difference between the 30-mg group and the controls was significant at PostnatalDay 22 (p < 0.05).

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462 LILIENTHAL ET AL.

10

2o6-33O•

1st day2nd day

•-I

4 5 6 7 8

[blocks of Mate]

10

e

8

.7

•6

5

4

3

2

1

0

FIG. 2. Learning curves for active avoidance learning on 2 consecutive days; (circles) controls; (triangles)5-mg group; (squares) 30-mg group. The number of avoidance reactions is given for each block of 10 trials(means ± 95% CL). On the first day the difference between the 30-mg animals and both other groups wassignificant (p< 0.05).

increased in the 30-mg group (F(2,33) = 3.89;p < 0.05) as shown in Fig. 3.

Operant Conditioning

In order to evaluate the performance onthe FI-30-sec schedule the whole interval wassplit into three parts of 10-sec duration each,and the average daily sum of reactions emit-ted by the rats was calculated across all 25 ses-sions. The resulting response distribution isshown in Fig. 4. Responding levels areslightly raised in the 30-mg group in all threeparts of the interval. The difference betweencontrols and the 30-mg group was marginallysignificant (randomization test, p < 0.1) inthe first 10 sec following the reinforcement.In the third part of the 30-sec interval thedifference in responding levels between the30-mg and the 5-mg group approached sig-nificance (p < 0.1). As can be seen in Fig. 4five of six control animals exhibited a contin-uous increase in reactions during the interval,whereas only three of six in the 5-mg groupand two of six in the 30-mg group did. Theindex of curvature failed to reveal significant

differences between groups, probably becauseof the difficulties in describing a curve by anarea index. A more recent approach to thisproblem is to fit spline functions to the re-sponse curve. This method has been success-fully used in analyzing serial response times(Bloxom, 1979). The daily response curveswere fitted to cubic regular splines and thelinear, quadratic, and cubic coefficients werecompared by analysis of variance with re-peated measures on the day factor. Therewere significant overall differences betweengroups for the linear coefficients (7*1(2,15)= 3.97;/7<.O5).

DISCUSSION

The exposure conditions chosen did notcause any weight reductions in the dams orin the offspring at any stage of development.Adipose tissue concentrations in the 5-mggroup are in the range of human values(DFG, 1988), despite the fact that the intakeneeded to achieve these concentrations ismuch higher in the rat. Litter size and weightwere not reduced either. However, the liver

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PRE- AND POSTNATAL PCB EXPOSURE 463

1.0

.7-

.6-

.5-

.2-

control 5 mg 30 mg

FIG. 3. Number of intertrial crossings in the shuttlebox divided by trials-to-criterion (means ± 95% CL).There were significant differences between 30-mg ratsand both other groups (p < 0.05).

to body weight ratio was increased in exposedsubjects. Locomotor activity in the open fieldwas elevated in the 30-mg group at PND 22,but not at PND 120. In monkeys PCBs arereported to enhance activity levels at the earlydevelopmental stages, whereas reducingthem in later phases (Bowman and Heironi-mus, 1981). On the other hand, long persist-ing increases in locomotor activity up to 1year after prenatal exposure have been de-scribed in mice (Agrawal et al., 1981) whichmay be related to the use of the highly toxiccoplanar 3,3',4,4'-tetraCB.

At about PND 70 active avoidance reac-tions were found to be increased in the 30-mggroup on the first day of training. Due to thesubsequent improved performance of thecontrol and 5-mg group this difference was nolonger detected on the second day. This resultis most probably a consequence of the higheractivity level in the higher exposed animalssince running facilitates successful perfor-mance in this type of learning. Similar effectson active avoidance were formerly reported

for amphetamine and other CNS stimulatingcompounds (Boff et al., 1982). This interpre-tation is further supported by the number ofintertrial responses which were also found tobe raised in the 30-mg group. In anotherstudy in rats, impaired active avoidance ac-quisition has been detected after PCB expo-sure (Pantaleoni et al., 1988). Since no mea-sures of internal exposure were given and be-cause of different treatment conditions andbehavioral test procedures, it is difficult tocompare that result with ours. However,there were lower activity levels in PCB-treated animals during and at the end ofweaning. In two other reports in mice (Stormet al., 1981; Tilson et al., 1979) no effect wasfound on the number of successful avoidanceresponses, but there were PCB-related in-creases in avoidance latencies. In the latter in-vestigation one-way instead of two-way

eoo-

S5OO-

!4O0-

J3O0-

200

1000

Controls 5 mg PC8

600- 600

500- 500"

400

300

200-

100

3 1 2 3Intervals of 10 seconds

30mgPC8

FIG. 4. Average daily sum of reactions on the FI 30-sec schedule for each 10-sec interval (means ± 95% CL);numbers 1-6 refer to individual animals in each group.The 30-mg group had elevated response levels in com-pared to those of the controls in the first 10 sec after thereinforcement (p < 0.1), and compared to those of the 5-mg group in the last 10 sec of the interval (p < 0.1). Notethe different reaction patterns with more u-shaped curvesfor the 30-mg group in contrast to the steadily increasingcurves for nearly all control animals.

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464 LILIENTHAL ET AL.

avoidance was studied, while in the former alower intensity of foot shock and a shorter in-tertrial interval were used. This suggests thatthe stimulus conditions and contingencies inour experiment facilitated avoidance re-sponding mediated by a higher activity level.Recent results from our laboratory on behav-ioral effects of treatment with lead or PCBsindicate that nonassociative factors like activ-ity and reactivity dominate AAL at the lowshock intensities (0.5 mA) used here, while athigher shock levels (1.0 mA) stimulus controlbecomes more effective (Bonatz et al., 1990;Lilienthal and Winneke 1989).

At PND 380 operant conditioning on a FI-30-sec schedule was employed as the third be-havioral paradigm. This particular schedulewas chosen because the number of reinforce-ments is not dependent on the number of re-sponses emitted. Thus, the rat is "free" topress the bar at any convenient rate. Therewere slightly more reactions emitted by the30-mg animals than by those in both othergroups, particularly during the first 10 sec af-ter the reinforcement when the delay to thenext reward is longest. This suggests a lack ofresponse inhibition. More remarkable, how-ever, were changes in the temporal pattern ofreactions as revealed by analysis with splinefunctions. Thus, taken together with the re-sults of locomotor testing in the open fieldand active avoidance conditioning, whichshow influences in the same direction, thereis a strong indication for a consistentalteration of activity-dependent behaviors.Changes in operant behavior on a FI schedulewere also reported in monkeys (Mele et al.,1986). The failure to detect an elevation ofactivity levels in open-field performance atPND 120 does not argue against this conclu-sion since differences are more likely to be-come apparent under more demanding be-havioral tasks such as AAL and operant con-ditioning.

Because a technical mixture of PCBs wasused a possible contribution of PCDFs has tobe considered. The analysis of our mixture re-vealed a content of about 2.5 mg PCDF/kg

PCB. This may have had an additional effect,but the most recent concern is about coplanarcongeners of PCBs themselves (Tanabe,1988). Of these, 3,3',4,4',5-pentaCB is knownto induce the microsomal oxidase system inthe liver at about the same level as PCDFsand not much less than 2,3,7,8-TCDD (Saw-yer and Safe, 1982; Safe, 1986). In commer-cial mixtures (Tanabe, 1988) and in humantissues (Kannan et ai, 1988) these coplanarPCB congeners are much more concentratedthan PCDFs and PCDDs. Thus, the toxicequivalent based on the calculation of induc-tion potency and concentration is higher forcoplanar PCB congeners than for the con-taminants of technical mixtures. The concen-.tration of the coplanar congener 3,3',4,4'-tet-raCB in the PCB mixture used was 1000 mg/kg total PCBs; 3,3',4,4',5-pentaCB and3,3',4,4',5,5'-hexaCB were not detected (de-tection limit 10 mg/kg total PCBs). However,other PCB congeners are known to interactwith coplanar PCBs resulting in overadditiveinduction (Parkinson and Safe, 1987). There-fore, a contribution of noncoplanar conge-ners to the observed effects should not be ne-glected.

The mechanism by which PCBs interferewith neuronal development and function re-sulting in behavioral alterations is still un-known. In neonatal mice a decreased densityof muscarinic cholinergic receptors in thehippocampus was found (Eriksson, 1988).Higher activity levels in exposed rats havebeen related to decreases in striatal dopaminelevels and receptors (Agrawal et ai, 1981).Effects on thyroid hormone receptors are dis-cussed as a possible mechanism for certainsigns of neuromuscular weakness, hypore-flexia, and hypotonicity in human babies(Rogan et ai, 1986b). There is some supportfor this by experimental studies in animals(Collins and Capen, 1980; Rickenbacher etai, 1986; Byrnes al., 1987;McKinney#a/.,1987). But other hormonal systems like theestrogen one may also be affected (Lundkvistet al., 1987). Recently, the role of estrogen re-ceptor modulation has been pointed out in

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PRE- AND POSTNATAL PCB EXPOSURE 465

the toxicity of 2,3,7,8-TCDD (Umbreit andGallo, 1988) and due to steric similarities ofthe compounds the assumption of an inter-ference of some PCB congeners with the ac-tions of estrogen and other steroids is reason-able.

The present study was designed to mimicthe conditions of human environmental ex-posure in an animal model. The exposurestarted before conception, extended through-out the periods of gestation and lactation inthe dams, and was continued for the offspringafter weaning. This implies that critical expo-sure periods as well as male-mediated effectscannot be identified in this study. From stud-ies of transfer rates of PCBs during gestationand lactation in rodents it is known that pre-natal transfer is about two to three orders ofmagnitude less than postnatal transfer (Ma-sudaetai, 1978;Takagi#a/., 1986). Internalexposure in the brain of weanling rats is in-creased by a factor of 3.5 in comparison tonewborns (Shain et ai, 1986).

Despite the low transfer of higher chlori-nated PCBs through placental barriers in hu-mans (Ando et ai, 1986), children born tomothers suffering from Yucheng exhibit neu-ropsychological deficits due to prenatal expo-sure even beyond the age of 7 years (Rogan etai, 1988). At environmental exposure levelsPCB-induced effects on development wereassociated with prenatal exposure in uterorather than with postnatal intake of humanmilk (Gladen et ai, 1988; Jacobson et ai,1985). The topic of critical developmental pe-riods for PCB exposure will be addressed in aforthcoming report.

ACKNOWLEDGMENTS

The authors thank Prof. Dr. Krauth for helpful adviceon the use of spline functions, Hannelore Krull for expertpreparation of the figures, and Andrea Lauterbach andMichael Lieverz for excellent technical assistance.

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