Toxicology Letters 224 (2014) 32–39

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Dopamine-dependent behavior in adult rats after perinatal exposure to purity-controlled polychlorinated biphenyl congeners (PCB52 and PCB180) Hellmuth Lilienthal a,∗ , Päivi Heikkinen b , Patrik L. Andersson d , Leo T.M. van der Ven e , Matti Viluksela b,c a Center of Toxicology, IPA – Institute for Prevention and Occupational Medicine, German Social Accident Insurance, Ruhr University of Bochum, Bürkle de la Camp Platz 1, 44789 Bochum, Germany b Department of Environmental Health, THL – National Institute for Health and Welfare, 70701 Kuopio, Finland c Department of Environmental Sciences, University of Eastern Finland, 70211 Kuopio, Finland d Department of Chemistry, Umeå University, 901 87 Umeå, Sweden e Center for Health Protection, RIVM - National Institute of Public Health and the Environment, 3720 BA Bilthoven, The Netherlands

h i g h l i g h t s • • • •

Developmental exposure to purity-controlled polychlorinated biphenyls affects cataleptic behavior in adult rats. PCB52 increased latencies to movement onset in female offspring. PCB180 decreased latencies to movement onset in male offspring. Benchmark dose analyses revealed that effects occur at moderate exposure levels.

a r t i c l e

i n f o

Article history: Received 18 June 2013 Received in revised form 15 October 2013 Accepted 16 October 2013 Available online 24 October 2013 Keywords: Polychlorinated biphenyls Dopamine Catalepsy Behavior Development Rats

a b s t r a c t Since knowledge about toxic effects of non-dioxinlike (NDL) PCBs is fragmentary, regulatory panels have concluded that risk assessment of these congeners is hampered or impossible. As the dopaminergic system is one of the main targets in PCB-related neurotoxic effects after developmental exposure, we selected catalepsy induced by the dopamine receptor blocker haloperidol to characterize effects of the NDL congeners PCB52 and PCB180 in adult offspring from exposed rat dams. Rat dams were treated with PCB congeners by gavage using six dose levels (total doses: PCB52 – 0, 30, 100, 300, 1000 or 3000 mg/kg body wt.; PCB180 – 0, 10, 30, 100, 300, or 1000 mg/kg body wt.) to allow benchmark dose analysis of the results. Testing of adult offspring (starting at 180 days of age) for catalepsy induced by injection with haloperidol revealed slightly prolonged latencies to movement onset in female offspring exposed to PCB52. Exposure to PCB180 resulted in more pronounced effects, with generally reduced latencies in male offspring. These results indicate reduced dopaminergic activity after PCB52 exposure, whereas the outcome for PCB180 may be related to increased extracellular dopamine as reported in the literature. Benchmark dose analyses revealed that both PCB congeners exerted effects mainly at moderate exposure levels. Together, these results underline the importance of effects on the dopaminergic system as indicated by studies in human females after occupational PCB exposure. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction 1.1. General

∗ Corresponding author. E-mail addresses: [email protected] (H. Lilienthal), [email protected] (P.L. Andersson), [email protected] (L.T.M. van der Ven), matti.viluksela@thl.fi (M. Viluksela). 0378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.10.016

Polychlorinated biphenyls (PCBs) are a group of industrial chemicals which were used in many devices and applications, including electrical capacitors and transformers, lubricants, and sealants in buildings. Their phase-out started early in the 1970s, with the prohibition of open use in several countries, followed by the termination of production and use in closed systems in the 1980s

H. Lilienthal et al. / Toxicology Letters 224 (2014) 32–39

and 1990s. There is a world-wide ban for PCBs since 2001 (e.g. review in Weber et al., 2011). Despite these regulations, PCBs leak from landfills and other diffuse sources to environmental compartments, due to their low chemical and biological degradation (Weber et al., 2011). Recently, unintended contamination was detected in air and water, due to the use of pigments containing PCBs formed during manufacturing processes (Hu et al., 2008; Hu and Hornbuckle, 2010). These factors lead to continued exposure of wild-life and humans (e.g. reviews in Ulaszewska et al., 2011; Consonni et al., 2012). Of the 209 congeners, some lack substitution at ortho-positions and can bind to the Ah receptor, leading to dioxinlike (DL) effects (e.g. review in White and Birnbaum, 2009). In contrast, most ortho-substituted PCB congeners do not show Ah receptor-mediated effects. These non-dioxinlike (NDL) PCBs result in endocrine (Hamers et al., 2011) and neurotoxic effects in vitro and in vivo (e.g. Fisher et al., 1998; Giesy and Kannan, 1998). Neurotoxicity was also reported for Ah receptor active compounds (e.g. review in Faroon et al., 2000), thus, demonstrating that both DL and NDL chemicals may influence neuronal function, albeit by different mechanisms. It was early recognized that PCB exposure in early developmental phases causes long-lasting effects on neural function and behavior (e.g. review in Seegal, 1996). 1.2. Dopaminergic neurochemical and neurobehavioral effects One of the main targets in the nervous system known to be affected by PCBs is the dopaminergic system. Early investigations found decreased dopamine (DA) content in cultured neurons (Seegal et al., 1989) as well as influences on DA content and metabolism in different brain regions (Seegal et al., 1986). Other dopaminergic effects detected in vivo and in vitro include influences on DA release in neuronal cultures (Angus and Contreras, 1996) and tissue preparations (Chishti et al., 1996). These effects are supposed to be mediated by action on the dopamine transporter (DAT) and the vesicular monoamine transporter (VMAT) (review in e.g. Fonnum and Mariussen, 2009). Recently, effects on the dopamine transporter were found in women after occupational exposure to PCBs (Seegal et al., 2010). Alteration of DA levels and function in the brain are likely to cause behavioral changes in exposed animals and humans. Increased locomotor activity was reported after subacute exposure to Aroclor 1254 in mice, in addition to oxidative damage and death of dopaminergic cells in the midbrain (Lee et al., 2012). Effects on locomotor activity are among the most frequently described changes induced by exposure to PCB mixtures and single congeners (e.g. review in Roegge and Schantz, 2006). Other studies examined the implication of dopaminergic function in PCB-induced behavioral effects, using the drug discrimination paradigm by which the stimulus properties of agonists and antagonists of DA receptors can be studied in PCB exposed animals. For instance, rat offspring exposed to a mixture of PCBs during development were trained to discriminate cocaine or amphetamine from saline in adulthood (Sable et al., 2011). Both drugs are well known to activate the DA system (e.g. Callahan et al., 1997; Nielsen et al., 1989). PCB exposed rats exhibited a higher percentage of responses to cocaine and a lower percentage to amphetamine in generalization tests, using lower doses than the training dose. The authors explained this outcome by differential influences on the DA transporter (Sable et al., 2011). An earlier report described the effect of in utero exposure to the coplanar PCB77 (3,3 ,4,4 -tetraCB) on the discrimination of apomorphine from saline at a dose selective for the D2 receptor (Lilienthal et al., 1997). Generalization tests with lower doses of the training drug and substitution tests with other drugs selective for D2 , D1 , 5-HT1A and GABA receptors failed to demonstrate any PCB-related effect on D2 or GABA mediated responses, whereas some signs for an implication of a D1 mediated influence and, in

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particular, for the interaction between serotonergic and dopaminergic responses were present. 1.3. Differential effects on the dopaminergic system Differential effects of different PCB congeners and mixtures on DA related function were frequently reported. Remarkably, ortho-chlorinated PCB congeners caused reduction of DA content in vitro and in vivo (Seegal et al., 1990, 1997), whereas elevated DA concentrations were observed after developmental exposure to Aroclor 1016 (Seegal, 1994) or to the coplanar congener PCB77 in rats, the latter causing consistent increases in the prefrontal cortex (Roth-Härer et al., 2001; Seegal et al., 1997, 2005). Also, increased extracellular DA concentrations were detected in the nucleus accumbens following developmental exposure to the ortho-substituted PCB180 in rats, whereas the ortho-chlorinated congeners PCB52 and PCB138 were not effective (Boix et al., 2011). However, activation of metabotrophic glutamate receptors led to elevated extracellular DA in controls and in PCB52 exposed animals, while reduction was found after PCB180 exposure. PCB138 caused increases in females and decreases in male offspring (Boix et al., 2011). Moreover, differential effects on the DA transporters DAT and VMAT2 were noted after acute exposure to Aroclor 1016 and Aroclor 1260 in mice. Both mixtures decreased DAT expression in the striatum, but only Aroclor 1260 reduced VMAT2 (Richardson and Miller, 2004). Differential effects of individual congeners may also underlie effects on DA receptors. The coplanar PCB77 failed to alter D1 and D2 receptor-related binding in the prefrontal cortex, nucleus accumbens, and in the striatum after developmental exposure (Roth-Härer et al., 2001), whereas PCB153 reduced D1 receptor densities in the cerebral cortex and striatum of male, but not female rat offspring, together with increases in cortical D2 -related density and affinity in weanling males (Coccini et al., 2011). These neurochemical differences are also reflected in effects on dopamine-related behavior. In the study by Boix et al. (2011), no alteration of locomotor activity was found after exposure to PCB52. PCB138 reduced activity in male and female offspring, whereas PCB180 caused reductions only in male rats. Deficits in delayed spatial alternation – a task reported to depend on dopaminergic activity in the prefrontal cortex (Bubser and Schmidt, 1990) – were observed after developmental exposure to PCB28, PCB118, and PCB153 (Schantz et al., 1995). The effectiveness of PCB118 is interesting, as it is a mono-ortho PCB which has considerable DL properties and has been assigned a toxic equivalency factor (TEF; Van den Berg et al., 2006). However, PCB77, PCB126, and TCDD itself did not affect delayed spatial alternation (Schantz et al., 1996) and the same was shown for PCB95, a tri-ortho-substituted congener (Schantz et al., 1997). The use of catalepsy induced by the D2 receptor blocker haloperidol as a measure to study DA dependent behavior in PCB exposed rats demonstrated that the coplanar PCB77 prolonged latencies to movement onset, whereas the increases caused by the same dose of the ortho-chlorinated PCB47 missed significance (Hany et al., 1999a). Effects on catalepsy were also described after developmental exposure of rats to the brominated flame retardants pentabromodiphenyl ether 99 (PBDE99) and hexabromocyclododecane (HBCD), the former causing prolongations in male rats as did the technical mixture Aroclor 1254, which was used for comparison (Lilienthal et al., 2005). In contrast, HBCD exposure resulted in reduction of latencies in female rat offspring (Lilienthal et al., 2009). 1.4. Purpose of the present experiments It appears that both DL-PCBs and NDL-PCBs influence dopaminergic functions after developmental exposure. Whereas the effects of DL congeners are rather uniform, actions of NDL-PCBs are

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more heterogeneous showing distinct profiles in endocrine activity (Hamers et al., 2011) and different assays of in vitro screening for neurotoxicity (Stenberg et al., 2011). Despite these differences, only a limited number of NDL-PCBs was examined for neurotoxicity in vivo, so far, including effects on the dopaminergic system, as described above. Therefore, the aim of the present experiments was to study additional NDL-PCBs to obtain information on the potency of these congeners to influence dopamine-dependent behavior after developmental exposure in rats. For this purpose, PCB congeners were highly purified to remove contamination by Ah receptor-active compounds which even at levels of 10 ␮g WHOTEQ/g NDL-PCB may modify the outcome, according to recent evaluations of toxic responses (US-EPA, 2012). PCB52 (2,2 ,5,5 tetraCB) and PCB180 (2,2 ,3,4,4 ,5,5 -heptaCB) were selected for the present investigation, since they are frequently used as indicator congeners in human biomonitoring studies and in analyses of environmental samples (EFSA, 2005). Moreover, both are found in different clusters, according to in vitro screening for neuronal effects (Stenberg et al., 2011) and their neurotoxicity was examined by only very few studies in vivo (Boix et al., 2011; Eriksson and Fredriksson, 1996).

have shown in previous experiments that exposure of pups via milk is higher than prenatal exposure across the placenta, at least for PCBs with a long half-life, like PCB180 (Hany et al., 1999b; Kaya et al., 2002; Lilienthal et al., 2000). More details of breeding procedures and maintenance of the offspring will be described in a separate report, together with general reproductive results (Roos et al., in preparation). A group of 85 rats from the offspring (42 males, 43 females derived from 4 to 6 litters per group) exposed to PCB52 were transferred from THL to the animal house of IPA (Bochum, Germany) at the age of 80 days. In addition, a group of 79 rats (39 males and 40 females derived from 5 to 7 litters per group) from the offspring exposed to PCB180 were transferred to IPA at the same age. After arrival, rats were housed in same sex groups of four rats in standard Macrolon® cages (59 × 38 × 20 cm, L × W × H, respectively), with saw dust bedding (ssniff, Soest Germany). Laboratory diet (RM-H, ssniff, Soest, Germany) and water were supplied ad libitum. Other housing conditions were the same as at THL. Rats were given an adaptation period of four weeks before the beginning of neurobehavioral and physiological testing. Measurements started with determination of sweet preference (Lilienthal et al., 2013), followed by cataleptic behavior and, finally, recording of brainstem auditory evoked potentials as described earlier (Lilienthal et al., 2011). Time intervals of at least four weeks were interspersed between the end of one test and the beginning of the next to prevent any serial effect. All experimental procedures using rats were approved by the local German authorities according to German law (Landesamt für Natur, Umwelt und Verbraucherschutz, Nordrhein-Westfalen, Germany, AZ: 9.93.2.10.32.07.280). Breeding and exposure protocols used at THL were approved according to Finnish law by the Research Animal Use Committee, University of Kuopio, Finland (06-79/19.06.2006).

2. Methods

2.3. Catalepsy

2.1. Chemicals

Beginning at the age of 180 days, rats were tested for haloperidol-induced catalepsy. The procedures of testing are described in detail elsewhere (Meerts et al., 2004). Briefly, catalepsy was induced by an intraperitoneal injection with the dopamine receptor blocker haloperidol (0.3 mg/kg body wt). Thirty and 60 min after the injection rats were placed in three unusual body postures in the following sequence, (1) standing upright with both front paws on a horizontal bar 9 cm above the floor, (2) clinging on a steep grid, with an angle of 80◦ to ground floor, and (3) lying on the surface of a box with each leg put in a hole on the surface. Catalepsy was measured by determination of latencies to onset of the respective movements – descent from the bar, replacement of one paw on the grid, and retraction of the first foreleg and hindleg from the holes. The differentiation between retraction of a foreleg and a hindleg on the box was conducted since time for retraction of forelegs and hindlegs is supposed to reflect the extrapyramidal side-effects and the antipsychotic activity, respectively (Ellenbroek et al., 1987). When a rat failed to move in one of the postures, the measurement was terminated after 180 s after which the rat was moved into the next posture or – in the case of the box – put back into an individual cage where it stayed until the next measurement at 60 min. When all measurements were complete, rats were put in a single cage to allow recovery from haloperidol treatment overnight.

PCB52 and PCB180 were provided by Chiron (Trondheim, Norway). Chemical methods used for analysis of TEQ normalized impurity levels (WHO toxic equivalents, according to Van den Berg et al., 2006) followed the procedures reported in Danielsson et al. (2008). Briefly, the purity of NDL-PCBs was analyzed by dissolving 25 mg of the compound in hexane which was eluted with 280 ml of 1:1 n-hexane/dichloromethane on a 3 g charcoal carbon column (Andersson Fine Chemicals, USA) mixed with celite (7.9/92.1; VWR International, France). The column was turned upside down and eluted with 280 ml of toluene. The toluene fraction was analyzed with gas chromatography coupled with high resolution mass spectrometry to determine DL-PCBs and PCDD/Fs. These analyses confirmed that only trace levels of DL compounds were present in the purified NDL-PCB standards. Purity was determined to 90 days vs 0.9 to 3.4 days, respectively) (Öberg et al., 2002; Tanabe et al., 1981). Total doses were 0, 10, 30, 100, 300, or 1000 mg/kg body wt. (thus, 0, 2.5, 7.5, 25, 75, 250 mg/kg at each application). We

2.4. Statistical analyses A total number of 37 male and 38 female rats exposed to PCB52 were tested for catalepsy. In a few cases, two rats of the same sex were taken from the same litter. There were two cases in control females as well as four cases in males and three in females of the top dose group (3000 mg/kg). Thus, the litter-based numbers were 12 controls (6 males, 6 females), 12 from the 30 mg-group (6 males, 6 females), 12 from the 100 mg-group (6 males, 6 females), 12 from the 300 mg-group (6 males, 6 females), and 10 from the 1000 mg-group (5 males, 5 females), and 8 from the 3000 mg-group (4 males, 4 females). Also, 78 rats exposed to PCB180 were tested for catalepsy. Of these, two males were from the same litter in the top dose group (1000 mg/kg). In addition, two females were derived from another litter of the same group. Therefore, the litter-based numbers were 14 controls (7 males, 7 females), 13 rats from the 10 mg-group (7 males, 6 females), 13 from the 30 mg-group (7 males, 6 females), 13 from the 100 mg-group (6 males, 7 females), and 13 from the 300 mg-group (6 males, 7 females), and 10 from the 1000 mg-group (5 males, 5 females). The replications were preplanned and should ensure that values are robust in groups from which only a few litters were available. Since there were only few occurrences, means were taken across two pups of the same sex, when taken from the same litter, to render a litter-based statistics. For the analyses of data benchmark dose modelling was used to determine significant dose–response relations and the dose levels at which deviations from control values exceeded a criterion value, the critical benchmark response (CES – critical effect size). Procedures of benchmark dose calculations are described in detail elsewhere (Slob, 2002; Van der Ven et al., 2006). Briefly, the PROAST software was used to model dose–response relations (http://www.rivm.nl/en/LIBRARY/SCIENTIFIC/MODELS/PROAST). Observed latency data were fitted to the dose–response model giving the best fit at a significance level of ˛ < 0.05. The dose–response model that gave the best description of the data was selected from a hierarchical family of models, by testing the likelihood of incremental complexity (increasing numbers of model parameters), according to Slob (2002). The CES for changes in catalepsy was set to 20% based on assumptions of biological significance of deviations from control level. The selection of this criterion was based

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on the assumption that similar exposure conditions would require a likewise adjustment of treatment in patients medicated with dopaminergic drugs. The benchmark dose (CED – critical effect dose) and the lower 10% confidence limit (CEDL) were calculated from the model of the dose–response curve for a given parameter. To exclude results with a high statistical variation, a CED/CEDL ratio of ≤10 was used as a criterion of reliability. To examine the statistical significance of the dose–response relations with conventional statistics, results were evaluated by preplanned analyses of linear, quadratic and cubic contrasts with the general linear models (GLM) procedure, separately for each sex, following analysis of general sex differences as a within-litter factor. The SAS Statistical Software (version 9.2, SAS Institute, Cary, NC) was used for these analyses. The significance level of these trend analyses was set to ˛ < 0.05.

3. Results 3.1. Physical development Full details of general developmental effects of PCB52 and PCB180 will be given in a separate report (Roos et al., in preparation). Briefly, maternal body weights were not affected by PCB52. In addition, body weights and mortality as well as developmental milestones, like eye opening, tooth eruption, and markers of sexual development, were not altered in exposed offspring. Also, body weights did not differ between groups in adult offspring at the time of catalepsy testing (Table 1). Exposure to PCB180 induced slight reductions in maternal body weight at the top dose level (1000 mg/kg). Also, decreased body weights were transiently observed in pups from the same dose level in the first week after birth, but normalized, thereafter. Neonatal mortality was slightly increased in offspring at the two highest dose levels (300 and 1000 mg/kg). Exposure did not influence markers of general development, whereas sexual development was slightly delayed. Slightly increased body weights (approximately 10%) were seen in adult female offspring after exposure to PCB180 (Table 1), but these changes were not dose-dependent.

Fig. 1. Latencies to movement onset on the box in adult female offspring after development exposure to PCB52. (A) Foreleg retraction, (B) hindleg retraction, both determined 60 min after injection with haloperidol. Dose-related increases were significant according to benchmark analyses (p < 0.05). Corresponding benchmark doses and lower bounds are given in the text. Mean ± SEM, n = 4–6/group.

3.2. Catalepsy Since analysis of data indicated main effects of sex on latency results in PCB52 (p < 0.05) and PCB180 rats (p < 0.05), dose-related effects were analyzed separately for males and females. Developmental exposure to PCB52 exerted only small effects on cataleptic behavior in adult rat offspring (Fig. 1 and Table 2). No significant dose–response relations were detected in males by benchmark dose analyses. Also, trend analyses by calculation of linear or higher contrasts failed to reveal significant dose-related effects on latencies in male offspring (p > 0.1), with the exception of a dose-related prolongation of descent latency from the bar which missed statistical significance in analysis of linear trends (0.05 < p < 0.1). According to benchmark dose analyses, significant increases in latencies (p < 0.05) were only observed in females for foreleg and hindleg retraction on the box 60 min after injection with haloperidol (Fig. 1). Corresponding CED and CEDL values were 445 and 261 mg/kg, respectively, for the foreleg and 487 and 284 mg/kg, respectively, for the hindleg. In addition, trend analyses revealed dose-related increases in latencies of foreleg retraction on the box (p < 0.05). To examine the consistency of effects, the sum of latencies was calculated by adding latencies in all three testing situations, namely, removal of the second paw from the bar, replacement of one paw on the grid and retraction of one foreleg on the box at 30 min and 60 min. These sums of latencies were not significant in either sex (p > 0.1). In contrast to PCB52, pronounced effects on cataleptic behavior were observed in male offspring after exposure to PCB180 (Fig. 2 and Table 3). Males exhibited dose-dependent decreases in latencies to movement onset. Benchmark dose analyses revealed significant dose–response relations (p < 0.05) for descent latencies

at the bar at 60 min and retraction latencies of forelegs on the box at 30 and 60 min and hindlegs at 60 min. In addition, the sum of latencies obtained at the bar (2nd paw), grid, and box (foreleg) was significantly related to dose at 30 and 60 min, showing the consistency of the effects. CED values for different parameters varied between 5.9 and 289.8 mg/kg (Table 3). Only one parameter was affected in exposed females. Latencies for hindleg retraction on the box were prolonged with a corresponding CED of 275.4 mg/kg (Table 3). According to trend analyses by the calculation of contrasts, significant linear trends were obtained for all measures shown in Fig. 2 and in Table 3 (p < 0.05). 4. Discussion The results of this study indicate that developmental exposure to PCB52 leads to slight alteration in haloperidol-induced catalepsy in female offspring, while significant dose-related effects were not detected in male offspring. Females exhibited dose-related prolongations of latencies for the retraction of forelegs and hindlegs in one test situation. No significant effects were obtained with the other two test devices or when the sum of latencies in all three situations was analyzed to examine consistency of the effects. This outcome suggests weak effects on the dopaminergic system in females. A number of studies have shown that ortho-chlorinated congeners decrease dopamine content in the striatum and other parts of the brain after developmental exposure (e.g. Castoldi et al., 2006; Seegal et al., 1997). Also, an environmentally relevant mixture of PCBs reduced tissue DA levels in an organotypic coculture of the developing striatum

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H. Lilienthal et al. / Toxicology Letters 224 (2014) 32–39

Table 1 Body weights of rats at the time of testing for haloperidol-induced catalepsy [g]. Total dose [mg/kg] PCB52

0

30

100

300

1000

3000

Females Males

288 ± 25 471 ± 34

303 ± 10 483 ± 29

276 ± 11 470 ± 21

295 ± 20 469 ± 36

305 ± 14 492 ± 16

299 ± 22 500 ± 41

Total dose [mg/kg] PCB180

0

10

30

100

300

1000

Females Males

263 ± 21 470 ± 42

290 ± 12 475 ± 39

291 ± 20 448 ± 17

276 ± 12 477 ± 25

289 ± 32 481 ± 46

286 ± 22 444 ± 36

Mean ± SD; PCB52: females, n = 4–6/group; males n = 4–6/group; PCB180: females, n = 5–7/group; males n = 5–7/group. Differences in body weight were not significant in PCB52 females and males and in PCB180 males (p > 0.1). Body weight increases were significant in PCB180 females (p < 0.05).

Table 2 Latencies to movement onset [s] in female and male rats exposed to PCB52a . Total dose [mg/kg] Females Bar, 2nd paw Grid Sum of latenciesb Males Bar, 2nd pawc Grid Box, foreleg Box, hindleg Sum of latenciesb a b c

0

30

100

300

1000

3000

40.6 ± 16.4 59.1 ± 11.0 149.7 ± 45.9

73.5 ± 39.5 130.0 ± 30.0 257.5 ± 10.5

62.6 ± 21.0 53.4 ± 14.5 176.2 ± 40.4

79.5 ± 23.5 52.8 ± 9.6 233.7 ± 54.4

80.0 ± 27.7 74.0 ± 24.9 269.2 ± 34.7

88.0 ± 36.1 82.0 ± 23.7 304.5 ± 96.1

99.7 32.4 92.7 100.6 224.8

± ± ± ± ±

16.4 5.6 27.9 28.7 45.9

92.7 51.2 102.7 155.3 246.5

± ± ± ± ±

28.3 12.6 34.6 24.7 64.4

141.8 39.0 68.8 150.6 249.6

± ± ± ± ±

25.0 4.5 29.4 29.4 44.4

85.8 48.0 60.0 69.2 193.8

± ± ± ± ±

27.7 17.7 11.2 23.9 36.5

155.0 69.8 102.6 129.0 327.4

± ± ± ± ±

25.0 22.9 31.8 31.4 65.0

158.6 59.6 106.4 145.3 324.6

± ± ± ± ±

12.6 15.7 26.6 14.1 26.4

Values were determined 60 min after the injection with haloperidol. All measures were not significant (p > 0.1). Sum of latencies obtained at the bar (2nd paw), grid and box (foreleg). Mean ± SEM; n = 4–6/sex/group. (p > 0.05).

and ventral midbrain (Lyng et al., 2007). Since the induction of catalepsy by classical neuroleptic compounds, like haloperidol, is mediated by their blocking effect on D2 receptors in the striatum

(e.g. Farde et al., 1992), PCB-related reduction of DA concentrations may exacerbate the action of haloperidol, resulting in longer latencies to movement onset. Similar prolongations of latency

Fig. 2. Latencies to movement onset in adult male offspring after development exposure to PCB180. Determined 30 min after injection with haloperidol: (A) Hindleg retraction on the box, (B) sum of latencies obtained at the bar, on the grid and on the box. Determined 60 min after injection with haloperidol: (C) Descent latency from the bar, (D) sum of latencies obtained at the bar, on the grid and on the box. Dose-related decreases were significant according to benchmark analyses (p < 0.05). Corresponding benchmark doses and lower bounds are given in Table 3. Mean ± SEM, n = 5–7/group.

H. Lilienthal et al. / Toxicology Letters 224 (2014) 32–39 Table 3 Critical effect doses (CED) and lower bounds (CEDL) for latencies to movement onset in rats exposed to PCB180. CED [mg/kg]

CEDL [mg/kg]

Box, hindleg, 60 min*,**

Female offspring 275.4

152.7

Bar, 2nd paw, 60 min*,** Box, hindleg, 30 min*,** Box, foreleg, 60 min*,** Box, hindleg, 60 min*,** Sum of latencies, 30 min*,** Sum of latencies, 60 min*,**

Male offspring 5.9 260.5 44.9 191.0 289.9 46.2

1.8 144.8 18.7 126.6 184.9 12.4

n = 5–7/sex/group; CED – critical effect dose for a deviation of 20% from controls; CEDL – lower bound (10%) of the CED. * Significant according to benchmark analysis (p < 0.05). ** Significant according to trend analysis (p < 0.05).

to movement onset were found after developmental exposure to PBDE99 (Lilienthal et al., 2005) which is one of the major constituents of the commercial PBDE mixture DE-71. This mixture was also described to cause reduction of DA concentrations in striatal synaptosome preparations from neonatal rats (Dreiem et al., 2010). Therefore, the effects of PCB52 on catalepsy are likely to result from effects on dopaminergic function in the striatum by decreasing local DA levels. Interestingly, PCB52 was one of the major congeners detected in primate brains after acute exposure to a technical PCB mixture that reduced DA concentrations in the striatum, substantia nigra and hypothalamus (Seegal et al., 1990). However, PCB52 appears to have only a weak potency to affect cataleptic behavior, since the corresponding CED and CEDL values were in the upper and medium dose range, respectively. In contrast to PCB52, more pronounced effects were detected in rat offspring exposed to PCB180. This congener caused dosedependent reductions in latencies only in male offspring, whereas female behavior was not changed, with the exception of hindleg retraction on the box on which slight latency increases were found similar to PCB52 females. Effects were more expressed at 60 min which is in accordance with previous experiments, using other PCB congeners (Hany et al., 1999a). Reduced catalepsy observed in males may be due to enhanced metabolic degradation of haloperidol in exposed animals. After treatment with pentobarbital, decreased sleeping times were described in rodents exposed to technical PCB mixtures in relation to induction of hepatic enzymes (Simmons and McKee, 1992; Villeneuve et al., 1972). Different cytochrome P450-dependent enzymes are involved in the metabolism of haloperidol, with prominent roles attributed to CYP3A4 and CYP2D6 in human microsomes (Fang et al., 2001; Murray, 2006). Whereas male rats generally exhibited somewhat more pronounced effects compared to females on induction of metabolizing liver enzymes after subacute exposure to PCB180, induction of CYP3A1 was only slightly higher in males than in females (Roos et al., 2011). Thus, it seems to be difficult to relate our findings of reduced cataleptic behavior in exposed males to gender-related differences in haloperidol metabolism, even though long-lasting effects of developmental treatment with enzyme inducing xenobiotic compounds have been described (e.g. Fujita et al., 1995). Diminished efficiency of haloperidol to induce catalepsy may also be due to altered receptor sensitivity. However, no alterations of D2 receptor density and affinity were found in the striatum of male and female rats after prenatal and lactational exposure the NDL PCB153 and only transient effects were found in the cortex of weanling males (Coccini et al., 2011). A general similarity between PCB153 and PCB180 has been noted according to toxicity profiling in vitro (Hamers et al., 2011). Also, the technical PCB mixture Aroclor

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1254 failed to affect D2 receptors in striatum of female mice after developmental exposure (Tian et al., 2011). Alternatively, decreased latencies may result from enhanced dopaminergic activity in the striatum, due to accumulation of DA in the synaptic cleft by inhibition of DAT as found after acute application of psychostimulants, like MDMA (3,4-methylenedioxymethamphetamine) and other designer drugs (e.g. Bogen et al., 2003). Since DA is not inactivated by reuptake into synaptic endings, an enhanced receptor response to DA may attenuate the blocking effect by haloperidol as previously reported (Banjaw et al., 2003). However, these effects were observed after acute treatment with psychogenic compounds and do not readily explain long-lasting effects in adult animals after perinatal exposure. On the other hand, PCB180 elevated extracellular DA in the nucleus accumbens of rats after pre- and postnatal exposure (Boix et al., 2011), but it remains to be determined, if this effect occurs also in the dorsal striatum. Furthermore, PCB180 led to decreased locomotor activity in male offspring (Boix et al., 2011). This may indicate a particular vulnerability of motor behavior in male versus female rats due to PCB180, although the directions of effects in spontaneous locomotor activity and in haloperidol-induced catalepsy do not match. The total dose of PCB180 in the study by Boix et al. (2011) was 36 mg/kg body weight which is, thus, only slightly higher than the two lowest levels used in the present study (10 and 30 mg/kg body wt.). Also, this dose is close to CED and CEDL values determined in our study, measuring 46 mg/kg and 12 mg/kg body weight, respectively, for the sum of latencies in males. In the present study, the use of total doses of PCB congeners for calculation of benchmark doses was preferred because the long half-life of PCB180 which measures at least 81–90 days (Öberg et al., 2002; Tanabe et al., 1981). Therefore, exposure of the offspring exceeds the days of treatment, covering all periods of prenatal and postnatal neurodevelopment. In accordance with this, postnatal exposure to higher chlorinated PCBs via milk was higher than exposure via the placenta after prenatal treatment (Hany et al., 1999b; Kaya et al., 2002; Lilienthal et al., 2000). Since the half-life of PCB52 is shorter (0.9–3.4 days, according to Öberg et al. (2002) and Tanabe et al., 1981), this congener was given up to postnatal day 10 to ensure that exposure continues throughout neurodevelopment. The finding of differential effects of PCB 52 and PCB180 on cataleptic behavior in male and female rats may be tentatively related to their influences on estrogenic processes which are known to modulate dopaminergic function in the striatum (e.g. review in Morrissette et al., 2008). According to in vitro experiments, both PCB congeners alter the action of estradiol. PCB 52 inhibits sulfonation of estradiol (Hamers et al., 2011). In untreated mammals, concentration of sulfoconjugated estradiol is higher than the level of estradiol in fetal plasma and uptake of estradiol sulfate with subsequent deconjugation by steroid sulfatase was found in fetal brain (e.g. Winikor et al., 2011). Therefore, inhibition of estradiol sulfonation may alter the bioavailability of estradiol in fetal brain. In contrast, PCB180 has an antagonistic activity at the estradiol receptor (Hamers et al., 2011). Thus, gender-related effects may result from interaction with different estrogenic processes, leading to modulation of dopaminergic neurotransmission. However, the relevance of the in vitro data for neurotransmission in vivo after developmental exposure and the precise mechanisms involved remains to be elucidated. Compared with effects of PCB52 and PCB180 on auditory thresholds determined with brainstem auditory evoked responses in these rats, benchmark doses for catalepsy were higher by factors of 2–3 in PCB52 exposed female offspring (Lilienthal et al., 2011). In contrast, PCB180 did not cause effects on auditory measures in male offspring and only one moderate effect on thresholds at 4 kHz in female rats, the benchmark dose of which being approximately

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two-fold higher than the only effect seen on catalepsy in females, namely, hindleg retraction on the box. It is, thus, apparent that developmental exposure to PCB52 led to more pronounced effects on auditory function compared to PCB180, but developmental PCB180 caused stronger effects on catalepsy than PCB52, leading to the conclusion that these congeners exhibit different neurotoxic profiles. Similar conclusions can be drawn from investigations of dopaminergic and glutamatergic effects of these congeners on neurotransmission (Boix et al., 2011). Since rats in this study were exposed only to PCB180 or PCB52 which were devoid of dioxinlike impurities, a comparison with human exposure is difficult. However, our results demonstrate that the dopaminergic system is a target of NDL PCB congeners. The examination of further NDL PCBs for catalepsy and other neurobiological effects within the program ATHON, which addresses toxic effects of ultrapure NDL congeners, is expected to provide a more complete spectrum of actions of this class of PCBs. Other NDL PCBs and mixtures may lead to stronger effects on dopaminergic function. In addition, also DL PCBs affect dopamine concentrations and function after developmental exposure (Hany et al., 1999a,b; Roth-Härer et al., 2001; Seegal et al., 1997). Therefore, PCB-induced effects on the dopaminergic system in the brain cannot be excluded in human and wildlife exposure. Indeed, effects on DAT densities have been reported in women after occupational exposure (Seegal et al., 2010). Together, these results demonstrate that dopaminergic effects of PCBs need to be further examined to elucidate possible risks of given exposure scenarios and the mechanisms involved. Conflict of interest None. Acknowledgements This work was supported by a grant from the European Commission, project ATHON [contract number FOOD-CT-2005022923], coordinator Helen Håkansson, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden. The authors are solely responsible for the contents of this paper which does not necessarily represent the opinion of the European Commission. The authors wish to thank Conny Danielsson (Umeå University) for purification of the NDL-PCBs. The technical help of Janne Korkalainen and Ulla Naukkarinen (THL) as well as of Michael Lieverz, Sven Schuchardt and Peter Wieskämper (IPA) is gratefully acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.toxlet.2013.10.016. References Angus, W.G., Contreras, M.L., 1996. Effects of polychlorinated biphenyls on dopamine release from PC12 cells. Toxicol. Lett. 89, 191–199. Banjaw, M.Y., Mayerhorfer, A., Schmidt, W.J., 2003. Anticataleptic activity of cathinone and MDMA (ecstasy) upon acute and subchronic administration in rat. Synapse 49, 232–238. Bogen, I.L., Haug, K.H., Myhre, O., Fonnum, F., 2003. Short- and long-term effects of MDMA (“ecstasy”) on synaptosomal and vesicular uptake of neurotransmitters in vitro andex vivo. Neurochem. Int. 43, 393–400. Boix, J., Cauli, O., Leslie, H., Felipo, V., 2011. differential long-term effects of developmental exposure to polychlorinated biphenyls 52, 138 or 180 on motor activity and neurotransmission. Gender dependence and mechanisms involved. Neurochem. Int. 58, 69–77. Bubser, M., Schmidt, W., 1990. 6-Hydroxydopamine lesion of the rat prefrontal cortex increases locomotor activity, impairs acquisition of delayed alternation

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