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Long-term effects of environmentally relevant doses of 2,2',4,4',5,5' hexachlorobiphenyl (PCB153) on neurobehavioural development, health and spontaneous behaviour in maternally exposed mice

Long-term effects of environmentally relevant doses of 2,2',4,4',5,5' hexachlorobiphenyl (PCB153)... Background: Polychlorinated biphenyls (PCBs) are widespread in the environment, human food and breast milk. Seafood is known to contain nutrients beneficial for the normal development and function of the brain, but also contaminants such as PCBs which are neurotoxic. Exposure to non-coplanar PCBs during brain development can disrupt spontaneous behaviour in mice and lead to hyperactive behaviour. Humans are chronically exposed to the highest relative levels of organochlorines in early childhood during brain development, though usually at doses which do not give clinical symptoms of toxicity. This study aimed to elucidate the developmental and behavioural effects of 2,2’,4,4’,5,5’ hexachlorobiphenyl (PCB153) in mice, mimicking human exposure during gestation and lactation. Methods: Environmentally relevant doses of PCB153 were added to the experimental diets. Feed concentrations were approximately 0.5, 6.5, and 1500 μg PCB153/kg feed, representing a realistic and a worst case scenario of frequent consumption of contaminated fish. The study also investigated the effects of maternal nutrition, i.e. a standard rodent diet versus a high inclusion of salmon. Mice pups were examined for physical- and reflex development, sensorimotor function and spontaneous behaviour from five days after birth until weaning. A selection of pups were followed until 16 weeks of age and tested for open field behaviour and the acoustic startle response (ASR) with prepulse inhibition (PPI). Blood thyroid hormones and liver enzymes, blood lipids and PCB153 content in fat were examined at 16 weeks. Statistical analyses modelled the three way interactions of diet, PCB exposure and litter size on behaviour, using generalized linear models (GLM) and linear mixed effect models (LME). The litter was used as a random variable. Non-parametric tests were used for pair wise comparisons of biochemical analyses. Results: Litter size consistently influenced pup development and behaviour. Few lasting PCB153 related changes were observed, but results indicated effects on synchronization of physical development. Perinatal PCB153 exposure appeared to reduce habituation and cause aggression in males, though not statistically significant. Conclusions: Litter size and maternal diet influenced physical development and function more than PCB153 in perinatally exposed mouse pups and supports the developmental importance of maternal care and the social environment. * Correspondence: mha@nifes.no † Contributed equally National Institute of Nutrition and Seafood Research (NIFES), P.O. box 2029 Nordnes, N-5817, Bergen, Norway Full list of author information is available at the end of the article © 2011 Haave et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 2 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 animal models are rare [26,27]. One well conserved Background response in mammals which has been deemed appropri- Polychlorinated biphenyls (PCBs) were produced in large ate for assessment of neurological aberrations, and pos- quantities before their extensive ban in the 1970s and sibly extrapolation to humans, is the startle response early ‘80s. Their persistence to degradation and their glo- with prepulse inhibition (PPI) [28]. Few experimental bal dispersion by air and ocean currents have made them studies examine the detrimental effects of PCB concen- omni-present in the environment, including food and trations or exposure modes relevant to humans. Seafood breast milk [1-4]. Human PCB exposure is typically in is a rich source of many beneficial nutrients important the form of long term dietary intake of relatively low for normal brain development and thyroid function doses. The highest exposure to organochlorines occurs [29,30] as well as a major source of dietary contami- during the first years of life in breast-fed infants [1,2,4-6] nants for seafood consumers [31-33]. The general pub- which coincides with the period of rapid brain growth lic, and in particular pregnant women are advised to and maturation, also called the postnatal transition per- increase their intake of seafood and fatty fish due to its iod [7] or Brain Growth Spurt (BGS) [8]. This is the per- nutritional value [34-36]. The importance of nutrition iod which has been shown to be vulnerable to has also been considered in recent epidemiological stu- organohalogen exposure in animal models [9-14]. In dies where the cognitive functions have been examined utero exposure to PCBs has furthermore been linked to in children in relation to PCBs, certain nutrients and cognitive and behavioural impairment in humans [15], breast feeding [37]. Nutrients have also been found to and high levels of organochlorines in breast milk have protect against the effects of several environmental con- been related to reduced neurological optimality in neo- taminants by means of counteracting oxidative effects nates [16]. In animals, exposure of young mice to low [38], sequestration [39], stimulation of metabolism [40] doses of di-ortho PCB, caused increasing hyperactivity or by reducing the uptake of the contaminants [41,42]. and altered spontaneous behaviour with age [12,13]. Dietary selenium supplementation in the mother has Mechanisms of effect of PCB exposure on cognitive also been shown to ameliorate detrimental effects of development are not fully elucidated, and have been methylmercury in murine offspring [43]. This suggests found to include structural as well as hormonal changes, that the nutritional composition of the diet during such as reduced density of cholinergic and muscarinic gestation and lactation may affect the experienced toxic receptors [11,12,17], and thyroid disruption in highly exposure of both dam and offspring, and their ability to exposed animals and humans [18-20]. Several mechan- tolerate the exposure. isms of effect have been suggested for PCB related thyr- The potential developmental effects of PCBs relevant oid disruption leading to cognitive effects [21]. to human exposure need to be examined using relevant Animals exposed to di-ortho PCBs during BGS also doses and exposure models. The aim of this study was show behavioural patterns which have been compared to evaluate possible adverse developmental effects in to both attention-deficit/hyperactivity disorder (AD/HD) young mice exposed to PCB153, in an exposure model and the progress of Alzheimer’s disease [12]. The non- relevant for humans, and to investigate the influence of dioxin like (NDL) PCBs like PCB153 have traditionally maternal intake of potentially ameliorating seafood not been perceived as very toxic, since they do not act nutrients. This includes the gestational and lactational through the same pathway as dioxins. Investigations exposure of offspring via cord blood and breast milk show that the toxic effect of these PCBs is not linked to during the vulnerable stages of BGS. their dioxin-like properties (coplanarity) [22]. Non- coplanar PCBs and the similar PBDEs have been Methods observedtobeneurotoxicand causemembranedisrup- The experiment and the animal facilities were approved tion or behavioural changes whereas coplanar and by the National Animal Research Authority (FDU, Nor- dioxin-like compounds do not cause an effect [10,23,24]. way). The study conforms to the requirements of the This implies that also NDL congeners should be consid- Norwegian National Committee for Animal Welfare, ered in toxicological evaluations. The recognition of the which closely conforms to the European Convention for potential neurotoxic effects of NDL PCB congeners has the protection of Vertebrate animals used for Experi- lead to advice from the European Food Safety Authority mental and other Scientific Purposes (Council of Europe (EFSA) that the NDL PCBs should be lowered as much no. 123, Strasbourg 1985). as possible, and that they should be included in risk- benefit evaluations and monitoring programs to increase Nutrition consumer safety [25]. However, established test paradigms that can reliably The experimental diets were produced in house accord- extrapolate results from animals to humans, or among ingtothe AIN-93 GRodent dietto meet1995NRC Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 3 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 Rat/Mouse Reproduction, Gestation and Lactation transfer to pups is also a relevant route of exposure for Values (DYETs Inc. formulation #110800). In order to humans, and includes transfer of metabolites of the par- investigate the effects of nutrition, two sets of diets were ent compound from the dam. Exposure to potentially produced with similar contaminant exposure but differ- toxic metabolites would not be obtained by direct expo- ent nutritional composition. Thus one diet was pro- sure of the pups, as pups have limited metabolic capa- duced with and one without inclusion of Atlantic city [49]. salmon (Salmo salar; Additional file 1). Briefly, the casein-based diet used casein sodium salt (Sigma Animal model and housing AldrichInc.) as themainsourceofprotein,and soy- Mice are much used as a model for developmental neu- bean oil as the sole source of lipids. The fish-based diet rotoxicity [10-14,17,50-62]. used 15% (per weight) freeze dried Atlantic salmon 54 female BALB/c mice from Charles River Inc. (Ger- raised on vegetable feeds [44] which gave low levels of many) were housed in groups of three in a large rat environmental contaminants [45]. The salmon was as a cage (Eurostandard Type IV) evenly distributed within source of both protein and lipids to the fish diet, and racks to compensate for any environmental variation. the fish-based feeds were added casein and soy-bean oil Housing conditions were standardized to 25 ± 2°C, 55 ± to reach the desired concentrations of 17% protein and 5% relative humidity, and 12:12 hr light-cycle, lights on 10% fat. The same concentrations of protein and lipid during the day. To promote natural behaviour and alle- were obtained for all diets, analysed by accredited meth- viate stress in the animals, cages were equipped with the ods at NIFES (Additional file 2). following environmental enrichment: a transparent poly- carbonate mouse igloo with an activity wheel (Bio-Serv), Spiking and doses of PCB153 a “mouse loft” (Tecniplast), aspen chewing sticks and To mimic human exposure animals were exposed to dust-free “Sizzle-Nest” (Scanbur). Dams were acclimated PCB153 via ad libitum food intake, through diets spiked to control diets ad libitum for one week before mating, with PCB153 (Chiron AS, Norway). Dietary PCB con- and experimental feeds were given ad libitum during centrations were verified by Gas Chromatography/Mass mating, gestation and lactation (until PND19; Figure 1). Spectrometry (GC/MS) in SIM mode, performed at The first day of mating was denoted gestation day (GD) NIFES by accredited methods based on previous publi- 0. Mating was performed within one week, with one cations [46,47]. One fish and one casein diet were left male per three females. The males were rotated after unspiked for control, while PCB153 was added in high the first oestrus cycle in case of infertility in the males, or low concentrations to both fish and casein diets, pro- which has been observed previously (data not shown). On GD16 females were separated into single cages ducing a total of six diets: Casein Control, Casein Low PCB and Casein High PCB, Fish Control, Fish Low PCB (Eurostandard Type III H) in order to monitor each lit- and Fish High PCB. The spiked low dose diet aimed at ter separately. Cages were checked twice daily for litters a PCB153 concentration similar to the concentration from GD18. The day of birth was denoted PND0. Pups typically found in farmed Atlantic salmon from Norway were weighed and handled every third day from PND5 [48], where PCB153 is the most prevalent congener. The until PND18 (Figure 1, Table 1). exposure to PCB153 from the diets with the spiked high Most animals were sampled on PND19. For the high concentrations represented a worst case scenario with level PCB groups and the Fish Control group, one male repeated consumption of fish with extremely high levels and one female sibling per litter were weaned on of PCB153. The intake of food was monitored by weigh- PND21, and monitored until week 16. All pups from ing any uneaten food daily, and calculation of the dose low dose groups were sacrificed on PND19 due to a low per kg body weight (BW) was done for gestation and lactation. Calculated doses ingested by the dams in the high dose groups were similar to the doses previously shown to produce persistent changes in spontaneous behaviour after single exposure on Postnatal day (PND) Mating 10 [13]. Dams with 30 g body weight consuming a max- 1 week 1 week 2 weeks ~3 weeks week 4-16 Monitoring Acclimation Gestation Behavioural testing imum of 12 g feed with 1500 ngCB153/g feed during lactation would have a peak daily intake of PCB153 of 1.7 μmol/kgBW*day. Feed and thus contaminant intake Experimental feed with PCB153 Clean chow diet by the dams peaked around PND11, which coincides Figure 1 Experimental outline. The experiment was performed with the BGS in mice [8]. The estimated exposure of during 16 weeks. Mice offspring were treated and handled as each pup would be highly influenced by pup body indicated. GD = Gestation day, PND = Postnatal day. weight and number of pups per litter. The lactational Weaning: PND21 Arrival GD0 Birth: GD21/PND0 PND 5 PND 8 PND 11 PND 15 PND 18 week 16 Behavioural testing and sampling Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 4 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 Table 1 Observations of physical development and behavioural tests in murine offspring Physical markers of development PND5 PND8 PND11 PND15 PND18 Week 16 Weight ++ + + + + Freeing of pinnae ++ Fur development ++ Incisor eruption ++ + Eye opening ++ Behavioural testing PND5 PND8 PND11 PND15 PND18 Week 16 Grasping reflex ++ Righting reflex ++ Climb/hang test ++ + Auditory startle ++ + Visual placing + Cliff drop aversion + Forelimb strength ++ Open field ++ Startle reflex (ASR/PPI) + Behavioural tests were performed on all pups in a litter from postnatal day (PND) 5 to PND18 and at 16 weeks of age. +: Signifies that the test was performed that day. number of litters in the Casein Low PCB group. All ani- produces a sharp sound without visible movement. mals were marked for identification and tracking of Except for hang/climb all reflex tests were scored 0-1, developmental history. Pups were given water and stan- where “1” is the display of characteristic behaviour [43]. dard rodent diet in pellets (chow, 10% fat) ad libitum For the hang/climb-test, graded levels of hanging and after weaning. Providing clean chow during development climbing were scored. Climbing was defined as moving and successfully replacing at least one fore- and one would imply that any observable toxic effects must have been caused by the exposure during early development. hind limb. The recordings were scored such that: 0 = no Pups were weighed weekly and inspected daily from hang, 1 = 1-14 sec, 2 = 15-29 sec, 3 = 30-44 sec, 4 = 45- Monday to Friday. Aggressive males were separated into 59 sec, 5 = > 60 sec, 6 = successfully climbing, replacing individual cages. All males were separated one week two limbs, 7 = successfully replacing three limbs, and 8 before behavioural testing in week 16, to minimize the = successfully replacing all four limbs while climbing. differences in caging conditions and stress level before Additionally, pups were tested for visual placing and the final behavioural test. cliff aversion [7] after eye opening on PND15. Forelimb strength was measured for pups on PND15 and -18 by a Behavioural testing grip strength meter (San Diego Instruments Inc., San From PND5 to PND18 the pups were observed and Diego, USA), and on PND18 pups were tested for anxi- tested for physical markers of development, reflexes and ety and spontaneous behaviour in an open field. The motor-function (Table 1). Behavioural tests were done open field arena was a (42 × 42) cm black polyethylene at the same time of day for each age-group, in a dedi- arena with 25 cm high walls, enclosed by white sheets cated room with the same temperature and lighting as hanging from the ceiling, and lit by indirect lighting to the housing conditions. All pups in a litter were tested create a uniform environment and exclude visual cues. by a single experimenter who was blinded to the treat- For the data analysis the open-field arena was divided ment. Where possible, at least one pup was left in the into two zones, a periphery, defined as a 7 cm wide area cage at any time during testing to relieve maternal along the walls, and a centre zone defined as the (28 × stress. On PND5, -8 and -11 all pups were tested for 28) cm square in the centre. On PND18 the animal was behavioural reflexes, based on the Fox battery [7] with placed in the centre of the field and latency to leave the centre was measured. In week 16 the animals were modifications previously described [63]. The individual tests were chosen based on experience from previous placed in an adjacent chamber, allowed to acclimate for trials [43,64]. The following tests were performed as two minutes, before a door to the open field was opened described by Folven et al. [43]: righting, hind limb and latency to enter the open field was measured. On grasp, a combined hang/climb-test and auditory startle PND18 movement was recorded for three minutes and reflex, with a small modification for the startle reflex in week 16 for 10 minutes, using a digital camera and test which was tested using a metal “click-box” that the behavioural tracking software SMART (San Diego Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 5 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 Instruments Inc., USA). The tracking recorded the intensities has previously been shown to be from 40-60% latency to leave the start zone (PND18 only), latency to [66]. Based on suspected gender differences in the PPI enter the field (week16 only), total distance travelled, response a decision was made apriori to separate time spent resting, the number of entries into the cen- genders. tre, and the total time spent in centre and periphery, Shortly before the final sampling two males from respectively. Also recorded was the distance travelled Casein High PCB and one male from Casein Control along the perimeter (% of total distance), and speed of were scanned with Magnetic Resonance Imaging (MRI) as a preliminary check for macro-anatomical changes in movement in the perimeter. The arena was wiped with the brain. No apparent changes in ventricle size or gross 70% ethanol and water between trials. The test order was randomized with respect to treatment group, litter brain morphology following the repeated perinatal PCB- and individuals in the litter. The test was performed at a exposure were seen, and the remaining animals were consistent time of day. not scanned. Complex behaviours: prepulse inhibition Sampling The startle response is an unconditioned response to an At PND19 most pups were euthanized and the liver was audible, tactile or sensory stimulus. The response is plas- sampled and frozen at -20°C until PCB analysis. At 16 tic, in that it can be modulated depending on other exter- weeks of age, remaining animals were euthanized by nal cues and repeated stimuli. Prepulse inhibition (PPI) is exsanguinations. Blood was drawn from the heart with a a phenomenon where a weaker stimulus prior to the syringe and mixed with 10 μl heparin (2.02 units/μl), main stimulus attenuates the response to a subsequent immediately centrifuged at 4°C, 2500 rpm for 5 minutes startle response. This modulation of the startle response and the serum frozen at -80°C until biochemical analyses is highly conserved in mammals, and has been used and (within two months). Liver enzyme analyses were per- discussed as a cross-species measure of sensorimotor gat- formed on serum samples to assess liver damage. Blood ing [28,65]. Abnormalities in PPI have been linked to sugar levels and blood lipids were also analysed in order neuropsychiatric disorders and diseases like schizophre- to examine differences related to early toxic exposure nia, Alzheimer’sand Huntington’s disease [28,65]. At 16 with different maternal diets. Analyses were performed weeks of age, the animals were tested for acoustic startle using a MAXMAT PL multipurpose diagnostic analyzer response (ASR) with prepulse inhibition (PPI) using the system (MAXMAT S.A. Montpellier, France). The fol- “Startle box” (Med Associates Inc., USA). The mice were lowing kits from DIALAB GmbH: Alanine transferase/ placed in a grid floor animal holder in the sound-attenu- Glutamate Pyruvate Transaminase (ALAT) with Pyri- ating cubicle, acclimated for five minutes with 60 decibel doxal 5’ phosphate, Alkaline Phosphatase (ALP), total (dB) ambient white noise and thereafter exposed to 40 Cholesterol (Chol), Gamma-glutamyl transferase (GGT) acoustic stimuli (Additional file 3). All stimuli were pre- and High Density Lipoprotein cholesterol (HDL); and sented as 120 dB white noise signals over a 60 dB ambi- from MAXMAT: Glucose (Glc), Lactate Dehydrogenase ent white noise. Prepulses (73, 75, 80 or 85 dB) were (LAD), and Triglycerides (TG) were used for serum ana- presented in pseudorandom order 100 ms prior to the lyses. Analyses of ALAT and GGT failed in a number of 120 dB main stimulus (Additional file 3). Inter-trial inter- samples and were omitted from results. Two additional vals varied randomly from 10 to 20 seconds. The ASR samples were omitted from the analysis due to outlying was defined as the peak amplitude after the 120 dB main results caused by technical problems during sampling. stimulus, subtracted the peak amplitude caused by spon- Additionally, serum was analysed for free Triiodothyr- taneous activity for 200 ms prior to prepulse presenta- onine (fT3), free Thyrosine (fT4) by RIA and Thyroid tion. Mean PPI for each prepulse intensity was calculated Stimulating Hormone (TSH) by IRMA, according to the as percent reduction in mean startle response with pre- manufacturer’s specifications (DRG international, Inc). pulse (ASR-pp) compared to the startle response without Only one replicate was analysed per sample due to lim- prepulse (ASR-simple, Additional file 3) according to the ited amounts of serum. formula (ASR-pp/ASR-simple)*100-100.Habituation was Concentrations of PCB153 in pup livers on PND19 calculated from the differences in mean response of the and abdominal fat in week 16 was analysed by Gas five last ASR-simple compared to the first five ASR-sim- Chromatography/Mass Spectrometry (GC/MS) in SIM ple, according to the formula [(ASR block3-ASR block1)/ mode, based on the same methods as used to analyse ASR block1]*100-100. With these approaches, normal the feeds [46,47]. inhibition of the startle response and habituation produce high negative values, whereas lack of inhibition and habi- Statistical analyses tuation gives values close to zero, or positive values. PPI The statistical methods employed here keep the litter as in untreated male BALB/c with this range of prepulse the experimental and statistical unit. This is highly Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 6 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 recommended when working with multiparous species, analyses and for analysis of PPI. The non-parametric as pups from the same litter are more similar than pups Kruskal-Wallis test of independent samples, followed by from different litters [67,68]. Mann-Whitney U pair wise comparison were used for comparison of means. Due to small sample sizes the Statistical models “Exact significance-test” was used. Significance levels All analyses aimed to assess how the response character- were set at alpha = 0.05 for all analyses. Because of missing data, two litters were excluded istics were influenced by the independent factors “diet” from the analysis of eye-opening: One litter in Fish High (fish or casein), “PCB153 level” (control,low,high),and PCB with 7 pups on PND12 and one litter in Casein “litter size” (1-10 pups) and their interactions. Inclusion of the dam as a random factor kept the litter as the sta- High PCB with 7 pups on PND13. Two litters in Fish tistical unit, as recommended [67,68]]A Linear Mixed Low PCB with only one pup were excluded from com- Effect Model (LME) [69]was employed to model influ- parisons of feed intake. Females were excluded from the ences on body weight, weight gain week 4-16 (corrected Open Field analyses in week16 due to technical difficul- for autocorrelation), Hepatosomatic Index (HSI = Liver ties performing the test. Males that died before week 16 weight*100/body weight),forelimbgripstrength,total were excluded from comparisons of body weight from distance travelled in the field, latency to leave the start week 4-16. zone (PND18) and latency to enter the field (week 16 only). The LME procedure accounts for the pups being Results grouped by the dam. For the reflex responses (PND5- Comparability among groups and litters 18) a generalized linear model (GLM) with quasi-/bino- There were no consistent, exposure related group differ- mial distribution was employed. The GLM model ences in body weight or mean feed intake among dams applied the response from each pup as a trial for the (Table 2). Reproductive success and litter size was not dam. In this way information about the number of pups different among groups that produced viable litters. The tested (trials) and the number of successful scores in the Casein Control group did not produce any viable litters. litter was included. Inclusion of the litter size as an The cause for the reproductive failure is not know, but independent variable provided a statistical test for the may reflect possible external stressors the first few days significance of the litter effect [68]. Additionally, in this after birth, which by chance affected this group the model the dam was included as a random factor repre- most. Several viable litters were born in other treatment senting the litter, due to differences in maternal care. groups on the same day. All diets were found to contain For the Open Field and the hang/climb test, scores were 10% fat and 17% protein. The vitamin supplements used measured as percent of maximum and compared using were identical for all feeds. Analysed concentrations of a variant of this model (GLMM), similar to the LME PCB153 in feeds are given in Table 2. Feed intake by procedure, but allowing for binomially distributed dams increased from approximately3.5 g/dayduring responses [70]. All models adjusted for uneven variance early gestation to a maximum of approximately 12 g/day of the residuals (heteroscedascity). The model selections during lactation. The feed intake by dams during lacta- were obtained from backward elimination. Due to the tion was strongly correlated with litter size (p < 0.001; reproductive failure in the Casein Control group, a full Figure 2). The feed intake during lactation and hence analysis including all possible terms was not possible. the calculated intake of PCB was significantly higher for The biological signal was then assessed through two Fish High PCB than for Casein High PCB (Table 2). separate models instead; model I used the influence from and interaction between litter size, diet and two Analyses of PCB153 levels PCB (low or high) only, disregarding the control Concentrations of PCB153 in pup livers at PND19 groups due to the missing Casein Control. Model II reflected maternal dietary intake. Pup liver PCB153 con- investigated the two-way interaction of litter size and centrations (Mean ± SEM) were 2.1 ± 0.5 ng/g, 9.6 ± PCB153 level among the fish groups (Fish Control, Fish 0.4 ng/g, 15.6 ± 5.7 ng/g, 2200 ± 300 ng/g and 1800 ± Low PCB and Fish High PCB). Where comparison of 200 ng/g ww for Fish Control, Casein Low PCB, Fish single groups was warranted, Tukey HSD Post hoc test Low PCB, Casein High PCB and Fish High PCB was used. Pearson’s product-moment correlation test respectively. was used to test the correlation between body weight and litter size in pups. Growth and development The above statistical analyses were performed by the Pup body weight was monitored from PND5-18 (Figure 3). statistical software R [71]. SPSS 15.0 for windows (SPSS Body weight of males and females are reported separately Inc., Chicago, IL, USA) was used for the nonparametric from week4 to week16 (Figure 4). The Fish Control comparisons of reproductive success, biochemical group generally had a higher body weight than pups in Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 7 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 Table 2 Feed concentration, body weight, reproductive output and feed intake by reproducing dams fed PCB153 in a fish or a casein-based diet Diet PCB153 Initial BW Final BW Repr. Litter size Intake gestation Intake lactation Gestation dose Lactation dose (n) ng/g feed dams dams succ % (g/day) (g/day) (μg/kgBW*day) (μg/kgBW*day) Casein Control 0.25 ± 0.12 n.d n.d 0 n.d n.d n.d n.d n.d (0) Fish Control 0.50 ± 0.07 28.9 ± 0.5 30.4 ± 0.2 33 4.7(4-5) 3.6 ± 0.2 7.7 ± 0.4 0.05 ± 0.00 0.12 ± 0.00 (3) Casein Low PCB 6.53 ± 0.12 29.1 ± 0.5 31.1 ± 0.9 33 6.3 (5-9) 3.6 ± 0.1 8.3 ± 0.7 0.73 ± 0.06 1.72 ± 0.23 (3) Fish Low PCB 6.57 ± 0.67 29.6 ± 0.8 31.3 ± 0.6 78 7.0 (4-9) 3.6 ± 0.1 8.6 ± 0.5 0.69 ± 0.03 1.63 ± 0.11 (5) (7) (7) Casein High PCB 1400 ± 60 28.4 ± 0.9 31.3 ± 1.5 56 5.8 (4-8) 3.9 ± 0.2 8.1 ± 0.3* 170 ± 10 350 ± 20* (6) Fish High PCB 1500 ± 300 30.0 ± 1.0 29.6 ± 0.7 56 6.8 (4-10) 3.5 ± 0.2 9.2 ± 0.3* 160 ± 10 420 ± 20* (5) Data are presented as mean ± SEM. Number of litters per group in brackets. Four litters were born in the Casein Control group, but no pups lived until postnatal day 5. Initial BW = BW after acclimation, final BW = BW end of weaning. Repr. succ: = percent successful reproduction in relation to the number of mated females. Litter size is given as mean (min-max). Feed intake during gestation (GD0-21) and lactation (PND0-21) was based on daily monitoring of feed intake. Daily doses were calculated based on average feed intake in grams and PCB153 concentrations in feeds, rounded off to the nearest ten ng/g for the high levels. Two females with single pups in Fish Low PCB were excluded from the calculation of litter size, feed intake and dose during gestation and lactation, but were included for comparisons of initial and final body weight (BW) of reproducing dams. n.d: No data due to reproductive failure. *: Groups that share a symbol are significantly different (Student’s t-test: p < 0.05). the PCB exposed groups. This trend persisted from lacta- male and female body weight until adulthood (p < 0.05 tion until week16, but was significant only in week 16. and p < 0.001) respectively. The mean litter size of the Fish Control group was slightly less than the Casein High PCB and Fish High Developmental effects of litter size, maternal diet and PCB groups (Table 2). The pup weight gain during lacta- PCB exposure tion was mainly influenced by litter size (Figure 2) and In the present study, the litter size was a prominent and was not significantly affected by the protein source (fish recurring factor which affected the behaviour and devel- or casein) or the PCB level. opment in a predictable and consistent manner. Physical Females in the Fish Control group were significantly heavier than females in Casein High PCB (p < 0.01) at week16 after correction for litter size (Figure 4, left Fish Fish Control Control panel). LME (adjusted for repeated measurements) Fish Fish Low Low demonstrated that the initial litter size had an effect on 12.5 Fish Fish High High Casein Casein Low Low Casein Casein High High 16 10.0 Intake Lactation Intake = 0.69** R Gestation 7.5 R 5.0 = 0.51** R = 0.01 2.5 0246 8 10 12 05 10 Litter size (count) Litter size (count) 5 5 8 8 11 11 15 15 18 18 Figure 2 influence of litter size on pup body weight and Postnatal day maternal feed intake. Left panel: Negative correlation was observed between pup bodyweight and litter size. Right panel: Figure 3 Body weight gain in offspring during lactation.Body Positive correlation was observed between maternal feed intake weight was measured on Postnatal day 5, -8, -11, -15 and -18. Each during lactation and litter size. Feed intake during gestation was not point with error bars represents the mean body weight ± SEM per affected by litter size. **: p < 0.001. group, in pups of both genders. Body weight (g) Pup bodyweight (g) Feed intake, dam (g) Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 8 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 At PND18 males spent more time in the centre of the Females Males 40 40 Fish Fish Control Control open field, spent less time moving fast and travelled a Fish Fish High High Casein Casein High High shorter distance than females (all groups compared, p < 30 30 0.001, p < 0.05 and p < 0.05 respectively). No other gender effects were seen. For the remaining analyses 20 20 genders were therefore grouped. On PND18 the fish groups left the central starting 10 10 zone significantly faster, and spent more time moving fast than casein groups (p ≤ 0.01 and p < 0.05, respec- tively). A trend towards lower resting time in the fish 0 0 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 14 14 15 15 16 16 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 14 14 15 15 16 16 groups than the casein groups was also seen (p < 0.08), Age (weeks) Age (weeks) indicating higher reactivity in the fish-groups regardless Figure 4 Body weight gain in juvenile mice week 4 to 16.Each of PCB exposure. No differences were observed for total point with error bars represents the mean body weight ± SEM in distance travelled; time spent moving slow and fast, female (left panel) and male (right panel) BALB/c mice exposed to PCB153 during gestation and lactation. The data include animals time spent resting or permanence time in the centre or that survived until week16. the periphery. Open field in week 16 Similar toPND18,inweek 16 malesfromthe fish development, such as body weight, fur development on groups entered the open field significantly faster than PND5, incisor eruption on PND11 and eye opening on PND14 were negatively related to litter size, with con- the casein group (p < 0.01). No effects of PCB exposure sistent and significant effects or trends in the two dif- were seen on the latency to enter the field or other ferent factorial models (data not shown). Regarding parameters tested. reflex development, a higher litter size significantly reduced the success rate in the hind limb grasp test Acoustic startle response with prepulse inhibition in (PND5), the hang/climb test (all days) and the startle week16 reflex (PND11) in both factorial models (p < 0.05) PPI tests showed large variability within the groups in Results for the forelimb grip strength were ambiguous. both genders. The small sample size and large variance Statistical analyses by model I showed that a maternal precludes statistical analyses, and the PPI test would fish diet was related to higher body weights in pups on need to be repeated with larger groups for conclusive all days from PND5 to PND18 (p < 0.05) after correc- results, and to determine possible diet, PCB exposure or litter effects. Although there were no significant differ- tion for the effect of litter size. The higher body weight ences among groups or genders, certain aspects of the may be the reason why a maternal fish diet was also findings deserve mentioning. This study shows that related to more fur on PND5 (p < 0.05), and tended to among the males, both of the PCB exposed groups give more pups with erupted incisors on PND11 (p < showed consistently less prepulse inhibition than the 0.1) as well as more pups with a startle response on controls (Additional file 4), although not significantly PND15 (p < 0.1). different. The reduction in startle response in exposed PCB exposure was not significantly related to body males was less than previously seen for BALB/c [66]. weight, growth or behavioural development. However, However exposed groups were not significantly different the timing of incisor eruption and eye opening appeared from the control group, and conclusions cannot be less synchronized in the PCB exposed pups than the drawn. controls. Comparing to Fish Control, the PCB153 exposed groups had earlier incisor eruption than the control group, but by PND11 the Fish Control was the Habituation to the acoustic startle response only group with full development of the trait. Similarly, A reduced startle response in the last set of simple sti- pups from Fish Low PCB and Fish High PCB had muli compared to the first set of simple stimuli indicates started eye-opening on PND12, whereas the pups from habituation, which is the expected response to repeated the Fish Control group had not. By PND14 there was stimuli. Noticeably, in males, both of the PCB exposed no difference among groups. groups showed the opposite response to habituation (Figure 5). The mean startle response was in fact higher Spontaneous behaviour in the open field PND18 in the last block of stimuli than at the start, indicating The open field used for assessment of spontaneous increased fear in that group, and no adaptation to the behaviour, anxiety and reactivity at PND18, showed situation. The large individual variability and the small differences in spontaneous behaviour among genders. dataset render conclusions difficult. Bo Bod dy y w we eiig ght ht ( (g g) ) Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 9 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 Table 3 Group size, body weight and hepatosomatic Females Males index for male and female offspring Fish Control Gender Group N BW BW HSI PND21 Week16 Week16 Fish High female Fish Control 3 16.9 ± 0.5 29.9 ± 1.1 4.97 ± 0.14 Casein High 6 13.0 ± 1.1 25.5 ± 0.8 4.36 ± 0.10 PCB Casein High ab Fish High PCB 5 14.9 ± 1.8 25.9 ± 1.3 4.65 ± 0.20 -4 -40 0 -2 -20 0 0 0 20 20 40 40 -6 -60 0 -4 -40 0 -2 -20 0 0 0 20 20 40 40 60 60 male Fish Control 3 16.6 ± 1.0 33.0 ± 2.3 4.38 ± 0.20 Habituation (%) Casein High 4 14.0 ± 1.2 30.6 ± 1.1 4.87 ± 0.38 Figure 5 Habituation to the acoustic startle stimuli in week 16. PCB Reduction in startle response at the end compared to at the start Fish High PCB 4 17.6 ± 3.1 27.0 ± 1.8 4.65 ± 0.62 of the trial. The bars and the error bars represent the percent The animals were exposed to PCB153 throughout gestation and lactation, via habituation (mean ± SEM). A negative value indicates a normal maternal consumption of spiked feeds based on fish or casein. High dose habituation response, with a lower startle response at the end than feeds (Fish High PCB and Casein High PCB) contained ~1500 μgPCB153/kg at the start of the trial. Sample sizes were (females/males): Fish feed. Data are presented as mean ± SEM of offspring that survived to week16. Control: n = 3/3; Fish High: n = 4/5; Casein high, n = 6/4. HSI = Hepatosomatic index, BW = body weight, PND = Postnatal day. Groups with different superscript letters are significantly different, within the same gender (Mann-Whitney U, p < 0.01). General observations of behaviour in PCB exposed males In week 4-16 allfemalegroupsand themalecontrols Casein High PCB group only (p < 0.05). TSH was below had 100% survival. Mortality was only observed in PCB the level of quantification (0.2 pg/L) for all samples, so exposed males. By week 12 the males in the Casein comparisons could not be made. High PCB group had a survival of 67% (4/6) and by week 13 males in the Fish High PCB group had a survi- Discussion val of 83% (4/5; Additional file 5). No deaths were The study aimed to elucidate if a worst case scenario caused by male aggression. However, all males in the with high maternal PCB153 intake through fish con- Casein High PCB group were separated into single cages sumption during brain development can be expected to since they developed aggressive behaviour. give any observable behavioural or developmental effects. Maternal nutrition is important for offspring Biochemical analyses of PCB concentrations, blood lipids development, and fish is also a known source of many and liver enzymes week 16 beneficial nutrients. The possible ameliorating effect of a PCB153 was analysed in abdominal fatin week16to maternal high fish diet was hence compared to a similar elucidate if maternal diet during gestation and lactation intake of PCB153 in a diet without fish. had an effect on pup metabolism and lasting body bur- Early pup development and behaviour as well as adult dens of PCB153. After 12 weeks on a standard chow behaviour and health were assessed as toxic effects have diet, groups still had PCB153 levels that reflected the previously been shown to manifest with increasing age maternal PCB intake during gestation and lactation. [10-12]. PCB153 concentrations were, Fish Control: 11.4 ± 2.7 The observations of this study suggested that continu- ng/g ww, Casein High PCB: 7000 ± 600 ng/g ww and ous maternal exposure to the single PCB153 congener Fish High PCB: 7000 ± 700 ng/g ww (Mean ± SEM, during gestation and lactation had little effect on para- high level groups rounded off to the nearest 100 ng/g meters of physical and neurobehavioural development in ww). Serum contents of blood lipids, liver enzymes and the offspring, but that survival and behaviour in early glucose showed minor differences only in levels of TG adulthood might have been compromised. The study among females (Additional file 6). There were no differ- suggested that PCB153 exposure might have affected the ences in blood lipids, liver enzymes or glucose among synchronization of physical development, such as eye the males. For the females in week 16, the Fish Control opening and incisor eruption. This tendency to dis- group had the highest HSI (Table 3), which coincided turbed synchronization of development has previously with a higher body weight of females in the Fish Control been observed in rats [72]. Disturbances in teeth devel- group (Figure 3). opment has also been observed in children exposed to PCBs during the so-called Yusho disasters where PCB Thyroid status contaminated rice-bran oil was ingested [73,74]. Nutri- No differences in fT3, fT4 or the ratio fT4/fT3 were tional effects on pup development were also present as found among groups (Figure 6). Comparing the genders, trends; as pups of dams fed the fish diets had an accel- males generally had lower fT3 and higher ratio fT4/fT3 erated development of physical traits, increased auditory than females, which was statistically significant in the startle reflex, and were more active in the open field Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 10 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 the mortality of the juvenile offspring in males. How- ever, the biochemical and behavioural evidence of last- 20 20 ing adverse effects of perinatal PCB153 exposure in adult mice were scant, possibly as a cause of the low ** sample size. The biochemical and behavioural data from week 16 are thus inconclusive. 15 15 Nutritional aspects Considering the nutritional aspect of the study, all diets had the same levels of nutrients and fulfilled require- ments for vitamins, minerals, protein and lipids (Addi- 10 10 tional file 7). An effect of nutrition on physical female female development has previously been seen with selenium male male supplementation, which increased fur development in neonate mouse pups [43]. It is possible that the more 6 6 nutritionally complex fish diets, which contained both fish, casein, fish oil and soy-oil, provided a more optimal nutrient composition which was beneficial during early 5 5 development. However, without a demonstrable adverse effect of PCB153 any possible ameliorating effects of fish could not be ascertained. 4 4 Neurobehavioural tests Previous studies have documented the validity and effi- 3 3 ciency of the neurobehavioural tests used, and have detected neurobehavioural differences in mouse pups using a similar test battery [43,64]. The fact that subtle 2 2 changes related to litter size were consistently detected in a predictable manner underlines the sensitivity and utility of the statistical methods. The effect of litter size, 0.4 0.4 when present, always affected the results in the expected direction, i.e. larger litters produced smaller and less agile pups. This again supports that the statistical meth- ** 0.3 0.3 ods employed would detect subtle changes where these were in fact present. Thus, any effects of PCB153 larger than the effect of litter size would presumably be detected. 0.2 0.2 Animal model Interpretations of the open field data at weaning were ambiguous, and the results did not provide a good indi- 0.1 0.1 cation of effects on anxiety from diet or dose PCB. The Fish Control Fish High Casein High interpretation of open field data must be performed Figure 6 Thyroid hormones in serum at week16 in murine with care [75]. The ambiguous results could be influ- offspring. Hormone concentrations of free triiodothyronine (fT3, enced by the immature exploratory behaviour in pre- upper panel), free thyroxine (fT4, middle panel) and the ratio fT4/fT3 weanling mice [64], but may also reflect the nature of (lower panel) in pups exposed to PCB153 via gestation and lactation. The boxes show the median and the inter-quartile range. BALB/c mice used. BALB/c mice are phenotypically * indicates significant difference between genders (Mann-Whitney U neophobic [76], which may have reduced the degree of pair wise comparison, p < 0.05). exploration and range of behaviour displayed in the open field, thus limiting the possibilities to discern compared to casein fed pups. Neither diet nor PCB effects of exposure among groups. Selection of a model exposure greatly affected development until adulthood, with higher reactivity to the test, such as the less but the maternal fish diet may have caused increased anxious C57BL/6 might have improved the ability to body weight in pups. PCB exposure may have increased discern effects among groups. Fr Fre e e e T4 T4 p pm m o oll//ll fr fre ee eT T 4 4 /fr /fre ee e T T 3 3 Fr Fre e e eT3 T3 p pm m o oll//L L Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 11 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 Statistical methods were exposed to sufficiently high levels during the vul- Above all, this study highlights the importance of taking nerable phase to potentially produce effects. Analyses of litter size into consideration in statistical evaluations of tissue levels in pups showed that they had high levels of neurodevelopmental responses. The findings support PCB153 in liver and fat on PND19, indicating a high previous observations showing that the litter effect may exposure to this di-ortho PCB congener throughout the be large and should always be included as a confounding period of BGS. The concentrations of PCB in the brains factor or predictor of the responses [7,67,68]. Not taking of pups which showed aberrant behaviour in previous into account the effects of intra-litter correlation and the studies [17] were low at the time of behavioural testing. litter size, may give an erroneous impression of group The current results suggest, however, that the effects are differences that are in reality caused by differences in lit- greater with exposure to a single, high bolus dose ter size and maternal care [68]. Particularly in studies of around BGS than with continuous exposure or slow teratogenic effects, or after maternal exposure of pups, accumulation throughout gestation and lactation. the litter effect may be prominent. It is essential to keep Neurotoxicity of the di-ortho PCBs have been shown in mind that the rearing condition (litter size) and the to manifest with age [10,12,13], but in the present study, toxic exposure (dose) of each litter are mutually depen- no significant effects in cognitive function or sensorimo- dent and are defined by the maternal care provided by tor gating were seen. However, the trends to difference each individual dam. Including both litter size and dam in PPI, the observed increase in male aggression in the analyses is therefore essential to discriminate throughout the trial and a corresponding lack of habi- between the individual effects, and will also provide a sta- tuationinthe ASRtestof the PCB exposed groups, tistical measure on the magnitude of the litter effect. indicate an increased stress level or increased anxiety in If any effects of PCB153 were indeed present and of males. The lack of habituation in the ASR-test may also the same magnitude as the effect of litter size it would be interpreted as a lack of ability to comprehend and have been detected, even with sample sizes as low as adapt to the novel situation which the startle-box repre- three animals. The benefit of the factorial design is that sents. The study of ASR/PPI needs to be repeated with it artificially increases the sample size when groups are a larger sample size for conclusions regarding the obser- combined for investigation of the factor they have in vations of lower PPI in PCB153 exposed males. common (diet, exposure level or litter size). This means that even though sample size is low for some groups, Biochemical effects of PCB153 the total number of litters exposed to i.e. “high level” is In previous epidemiologic studies of thyroid hormone dis- the sum of Fish High PCB and Casein High PCB ruption, exposure to the background levels of persistent together, which is 5+6 = 11 litters. Thus the statistical organic pollutants (POPs) in Scandinavia had little effect power for detecting effects of a high dose PCB153 is on thyroid hormones in breast-fed infants [77]. In con- increased compared to using a one-way ANOVA where trast, severe thyroid hormone disruption and impacts on the groups are investigated separately. For the factor cognitive function were seen after consumption of highly “litter size” it means investigating all litters with the PCB contaminated rice-oil [73], potentially containing same number of pups, regardless of diet or dose, to see traces of furanes and unidentified contaminants as well as if they are more similar than litters of different sizes. PCB. A combination of mixture-effects and exposure The low sample size however obstructs detection of dif- levels is likely to be of importance to the thyroid disrupt- ferences among groups and the potentially ameliorating ing effects seen in other studies, whereas the PCB153 con- effects of maternal nutrition. Although reproductive out- gener alone at the present doses did not affect the thyroid put in this study was lower than expected, the differ- hormone levels or the blood parameters investigated. Only ences in reproduction appeared not to be related to a slight difference in TG levels was seen among females. PCB153 exposure. However, the TG levels were seemingly reduced by PCB exposure, which is in contrast to previous findings [78] Behavioural and cognitive effects of PCB153 where exposure to a mixture of POPs increased the levels It seems to be a recurring finding that organohalogen of TG in rats. Since the TG levels in males showed no exposure at low doses that do not affect growth or phy- consistent differences related to PCB153 exposure, the sical development, still produce aberrant behaviour at higher TG and also HSI in Fish Control females may be later stages [11-13,17,59]. As concerns the present expo- explained by the higher body weight sure level, the repeated doses to the dams were in the range of the single exposure doses (1.4 μmol/kgBW*day) Human relevance of the study regarding neonatal PCB that have been shown to produce aberrant behavioural exposure effects after a single oral dose on PND10 [12,13,56]. The findings imply that resource availability, nutrition, Thus it was assumed that pups in the present study maternal care and social environment during gestation Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 12 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 and early development affect development and beha- Additional file 7: Concentrations of nutrients in the experimental viour to a much larger extent than the present exposure feeds. Table of analysed concentrations of protein, fat and selected vitamins and fatty acids in the casein- and fish-based diets. to PCB153. In mice these parameters are defined by the litter size. This suggests that the developmental starting point may outweigh other influences. The importance of rearing conditions and nutrient on development and Acknowledgements The authors wish to express their gratitude to Aase Heltveit, Lars-Erik behaviour has recently been reviewed [58]. The impor- Pindard, Edel Erdal and Jacob Wessel at NIFES for technical assistance. tance of maternal care and nutrition also supports Thanks to Dr. Frits A. Thorsen at the Institute of Biomedicine, University of recent studies in children [37], where Docosahexaenoic Bergen, for performing the MRI, Gaute Velle at the University of Bergen for editing figures, and all other personnel at NIFES involved with feed- and Acid (DHA) and selenium, as well as breastfeeding were tissue analyses. found to protect against adverse effects of environmen- This work was funded by the EU-project “AQUAMAX"; EU-Contract no. tal exposure in early childhood [37]. Breastfeeding and 016249-2, and the National Institute of Nutrition and Seafood Research (NIFES). maternal care have also been suggested to modify the effects of contaminants in human behavioural develop- Author details ment [79]. National Institute of Nutrition and Seafood Research (NIFES), P.O. box 2029 Nordnes, N-5817, Bergen, Norway. Department of Biology, University of Bergen, P.O. box 7803, N-5020, Bergen, Norway. Department of Biological Conclusions and Medical Psychology, University of Bergen, Jonas Lies vei 91, N-5009, Exposure of mice offspring to the single congener Bergen, Norway. The Norwegian Forest and Landscape Institute, Fanaflaten 4, N-5244 Fana, Norway. PCB153 via the dam did not result in effects on physical development and behaviour that exceeded the biological Authors’ contributions influence of litter size. Synchronization of development MHA took active part in designing the project, preparing the experimental diets, animal housekeeping, collecting behavioural data, tissue sampling; such as eye opening and incisor eruption may have been performing the statistical analyses, writing the original draft and completing compromised by neonatal PCB exposure. A maternal the manuscript. ABE took active part in preparation of the experimental fish diet increased pup body weight and accelerated phy- diets, animal housekeeping, data collection and statistical analyses and performing the behavioural tests. FJE was involved in selection and design sical development, but did not alter pup reflex develop- of the ASR-PPI and the analysis of ASR-PPI-data. EHE selected, implemented ment or behaviour to a great extent. Although a larger and validated the statistical models using the R-software. TBR and AKL took sample size would increase the statistical power and part in designing the study and in revising and proofreading the manuscript. All authors read and approved the final manuscript. would help detect differences, it was shown that random variables, such as litter size have a great impact on pup Competing interests development and behaviour. This needs to be consid- The authors declare that they have no competing interests. ered in experimental design and statistical analyses. The Received: 1 September 2010 Accepted: 13 January 2011 study demonstrated that subtle differences can be Published: 13 January 2011 detected using the current methods and statistical treat- ment of the data. References 1. Guvenius DM, Aronsson A, Ekman-Ordeberg G, Bergman A, Noren K: Human prenatal and postnatal exposure to polybrominated diphenyl Additional material ethers, polychlorinated biphenyls, polychlorobiphenylols, and pentachlorophenol. Environ Health Perspect 2003, 111:1235-1241. 2. Noren K, Meironyte D: Certain organochlorine and organobromine Additional file 1: Composition of experimental diets. Table of dietary contaminants in Swedish human milk in perspective of past 20-30 years. components in the fish and casein based diets, given as g/kg feed. Chemosphere 2000, 40:1111-1123. 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Eriksson P, Fischer C, Fredriksson A: Polybrominated Diphenyl Ethers Cite this article as: Haave et al.: Long-term effects of environmentally (PBDEs), a Group of Brominated Flame Retardants, can Interact with PCB relevant doses of 2,2’,4,4’,5,5’ hexachlorobiphenyl (PCB153) on in Enhancing Developmental Neurobehavioral Defects. Toxicol Sci 2006. neurobehavioural development, health and spontaneous behaviour in 57. Eriksson P, Jakobsson E, Fredriksson A: Brominated flame retardants: a maternally exposed mice. Behavioral and Brain Functions 2011 7:3. novel class of developmental neurotoxicants in our environment? Environ Health Perspect 2001, 109:903-908. 58. Branchi I: The mouse communal nest: investigating the epigenetic influences of the early social environment on brain and behavior development. Neurosci Biobehav Rev 2009, 33:551-559. 59. Branchi I, Alleva E, Costa LG: Effects of perinatal exposure to a polybrominated diphenyl ether (PBDE 99) on mouse neurobehavioural development. 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R Development Core Team: R: A language and environment for statistical Submit your next manuscript to BioMed Central computing. R Foundation for Statistical Computing, Vienna, Austria; 2009. and take full advantage of: 72. Gralewicz S, Wiaderna D, Lutz P, Sitarek K: Neurobehavioural functions in adult progeny of rat mothers exposed to methylmercury or 2,2’, 4,4’, • Convenient online submission 5,5’-hexachlorobiphenyl (PCB 153) alone or their combination during gestation and lactation. Int J Occup Med Environ Health 2009, 22:277-291. • Thorough peer review 73. Masuda Y: Toxic effects of PCB/PCDF to human observed in Yusho and • No space constraints or color figure charges other poisonings. Fukuoka Igaku Zasshi 2009, 100:141-155. • Immediate publication on acceptance 74. Tilson HA, Jacobson JL, Rogan WJ: Polychlorinated biphenyls and the developing nervous system: cross-species comparisons. Neurotoxicol • Inclusion in PubMed, CAS, Scopus and Google Scholar Teratol 1990, 12:239-248. • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Behavioral and Brain Functions Springer Journals

Long-term effects of environmentally relevant doses of 2,2',4,4',5,5' hexachlorobiphenyl (PCB153) on neurobehavioural development, health and spontaneous behaviour in maternally exposed mice

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Springer Journals
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Copyright © 2011 by Haave et al; licensee BioMed Central Ltd.
Subject
Biomedicine; Neurosciences; Neurology; Behavioral Therapy; Psychiatry
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1744-9081
DOI
10.1186/1744-9081-7-3
pmid
21232145
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Abstract

Background: Polychlorinated biphenyls (PCBs) are widespread in the environment, human food and breast milk. Seafood is known to contain nutrients beneficial for the normal development and function of the brain, but also contaminants such as PCBs which are neurotoxic. Exposure to non-coplanar PCBs during brain development can disrupt spontaneous behaviour in mice and lead to hyperactive behaviour. Humans are chronically exposed to the highest relative levels of organochlorines in early childhood during brain development, though usually at doses which do not give clinical symptoms of toxicity. This study aimed to elucidate the developmental and behavioural effects of 2,2’,4,4’,5,5’ hexachlorobiphenyl (PCB153) in mice, mimicking human exposure during gestation and lactation. Methods: Environmentally relevant doses of PCB153 were added to the experimental diets. Feed concentrations were approximately 0.5, 6.5, and 1500 μg PCB153/kg feed, representing a realistic and a worst case scenario of frequent consumption of contaminated fish. The study also investigated the effects of maternal nutrition, i.e. a standard rodent diet versus a high inclusion of salmon. Mice pups were examined for physical- and reflex development, sensorimotor function and spontaneous behaviour from five days after birth until weaning. A selection of pups were followed until 16 weeks of age and tested for open field behaviour and the acoustic startle response (ASR) with prepulse inhibition (PPI). Blood thyroid hormones and liver enzymes, blood lipids and PCB153 content in fat were examined at 16 weeks. Statistical analyses modelled the three way interactions of diet, PCB exposure and litter size on behaviour, using generalized linear models (GLM) and linear mixed effect models (LME). The litter was used as a random variable. Non-parametric tests were used for pair wise comparisons of biochemical analyses. Results: Litter size consistently influenced pup development and behaviour. Few lasting PCB153 related changes were observed, but results indicated effects on synchronization of physical development. Perinatal PCB153 exposure appeared to reduce habituation and cause aggression in males, though not statistically significant. Conclusions: Litter size and maternal diet influenced physical development and function more than PCB153 in perinatally exposed mouse pups and supports the developmental importance of maternal care and the social environment. * Correspondence: mha@nifes.no † Contributed equally National Institute of Nutrition and Seafood Research (NIFES), P.O. box 2029 Nordnes, N-5817, Bergen, Norway Full list of author information is available at the end of the article © 2011 Haave et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 2 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 animal models are rare [26,27]. One well conserved Background response in mammals which has been deemed appropri- Polychlorinated biphenyls (PCBs) were produced in large ate for assessment of neurological aberrations, and pos- quantities before their extensive ban in the 1970s and sibly extrapolation to humans, is the startle response early ‘80s. Their persistence to degradation and their glo- with prepulse inhibition (PPI) [28]. Few experimental bal dispersion by air and ocean currents have made them studies examine the detrimental effects of PCB concen- omni-present in the environment, including food and trations or exposure modes relevant to humans. Seafood breast milk [1-4]. Human PCB exposure is typically in is a rich source of many beneficial nutrients important the form of long term dietary intake of relatively low for normal brain development and thyroid function doses. The highest exposure to organochlorines occurs [29,30] as well as a major source of dietary contami- during the first years of life in breast-fed infants [1,2,4-6] nants for seafood consumers [31-33]. The general pub- which coincides with the period of rapid brain growth lic, and in particular pregnant women are advised to and maturation, also called the postnatal transition per- increase their intake of seafood and fatty fish due to its iod [7] or Brain Growth Spurt (BGS) [8]. This is the per- nutritional value [34-36]. The importance of nutrition iod which has been shown to be vulnerable to has also been considered in recent epidemiological stu- organohalogen exposure in animal models [9-14]. In dies where the cognitive functions have been examined utero exposure to PCBs has furthermore been linked to in children in relation to PCBs, certain nutrients and cognitive and behavioural impairment in humans [15], breast feeding [37]. Nutrients have also been found to and high levels of organochlorines in breast milk have protect against the effects of several environmental con- been related to reduced neurological optimality in neo- taminants by means of counteracting oxidative effects nates [16]. In animals, exposure of young mice to low [38], sequestration [39], stimulation of metabolism [40] doses of di-ortho PCB, caused increasing hyperactivity or by reducing the uptake of the contaminants [41,42]. and altered spontaneous behaviour with age [12,13]. Dietary selenium supplementation in the mother has Mechanisms of effect of PCB exposure on cognitive also been shown to ameliorate detrimental effects of development are not fully elucidated, and have been methylmercury in murine offspring [43]. This suggests found to include structural as well as hormonal changes, that the nutritional composition of the diet during such as reduced density of cholinergic and muscarinic gestation and lactation may affect the experienced toxic receptors [11,12,17], and thyroid disruption in highly exposure of both dam and offspring, and their ability to exposed animals and humans [18-20]. Several mechan- tolerate the exposure. isms of effect have been suggested for PCB related thyr- The potential developmental effects of PCBs relevant oid disruption leading to cognitive effects [21]. to human exposure need to be examined using relevant Animals exposed to di-ortho PCBs during BGS also doses and exposure models. The aim of this study was show behavioural patterns which have been compared to evaluate possible adverse developmental effects in to both attention-deficit/hyperactivity disorder (AD/HD) young mice exposed to PCB153, in an exposure model and the progress of Alzheimer’s disease [12]. The non- relevant for humans, and to investigate the influence of dioxin like (NDL) PCBs like PCB153 have traditionally maternal intake of potentially ameliorating seafood not been perceived as very toxic, since they do not act nutrients. This includes the gestational and lactational through the same pathway as dioxins. Investigations exposure of offspring via cord blood and breast milk show that the toxic effect of these PCBs is not linked to during the vulnerable stages of BGS. their dioxin-like properties (coplanarity) [22]. Non- coplanar PCBs and the similar PBDEs have been Methods observedtobeneurotoxicand causemembranedisrup- The experiment and the animal facilities were approved tion or behavioural changes whereas coplanar and by the National Animal Research Authority (FDU, Nor- dioxin-like compounds do not cause an effect [10,23,24]. way). The study conforms to the requirements of the This implies that also NDL congeners should be consid- Norwegian National Committee for Animal Welfare, ered in toxicological evaluations. The recognition of the which closely conforms to the European Convention for potential neurotoxic effects of NDL PCB congeners has the protection of Vertebrate animals used for Experi- lead to advice from the European Food Safety Authority mental and other Scientific Purposes (Council of Europe (EFSA) that the NDL PCBs should be lowered as much no. 123, Strasbourg 1985). as possible, and that they should be included in risk- benefit evaluations and monitoring programs to increase Nutrition consumer safety [25]. However, established test paradigms that can reliably The experimental diets were produced in house accord- extrapolate results from animals to humans, or among ingtothe AIN-93 GRodent dietto meet1995NRC Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 3 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 Rat/Mouse Reproduction, Gestation and Lactation transfer to pups is also a relevant route of exposure for Values (DYETs Inc. formulation #110800). In order to humans, and includes transfer of metabolites of the par- investigate the effects of nutrition, two sets of diets were ent compound from the dam. Exposure to potentially produced with similar contaminant exposure but differ- toxic metabolites would not be obtained by direct expo- ent nutritional composition. Thus one diet was pro- sure of the pups, as pups have limited metabolic capa- duced with and one without inclusion of Atlantic city [49]. salmon (Salmo salar; Additional file 1). Briefly, the casein-based diet used casein sodium salt (Sigma Animal model and housing AldrichInc.) as themainsourceofprotein,and soy- Mice are much used as a model for developmental neu- bean oil as the sole source of lipids. The fish-based diet rotoxicity [10-14,17,50-62]. used 15% (per weight) freeze dried Atlantic salmon 54 female BALB/c mice from Charles River Inc. (Ger- raised on vegetable feeds [44] which gave low levels of many) were housed in groups of three in a large rat environmental contaminants [45]. The salmon was as a cage (Eurostandard Type IV) evenly distributed within source of both protein and lipids to the fish diet, and racks to compensate for any environmental variation. the fish-based feeds were added casein and soy-bean oil Housing conditions were standardized to 25 ± 2°C, 55 ± to reach the desired concentrations of 17% protein and 5% relative humidity, and 12:12 hr light-cycle, lights on 10% fat. The same concentrations of protein and lipid during the day. To promote natural behaviour and alle- were obtained for all diets, analysed by accredited meth- viate stress in the animals, cages were equipped with the ods at NIFES (Additional file 2). following environmental enrichment: a transparent poly- carbonate mouse igloo with an activity wheel (Bio-Serv), Spiking and doses of PCB153 a “mouse loft” (Tecniplast), aspen chewing sticks and To mimic human exposure animals were exposed to dust-free “Sizzle-Nest” (Scanbur). Dams were acclimated PCB153 via ad libitum food intake, through diets spiked to control diets ad libitum for one week before mating, with PCB153 (Chiron AS, Norway). Dietary PCB con- and experimental feeds were given ad libitum during centrations were verified by Gas Chromatography/Mass mating, gestation and lactation (until PND19; Figure 1). Spectrometry (GC/MS) in SIM mode, performed at The first day of mating was denoted gestation day (GD) NIFES by accredited methods based on previous publi- 0. Mating was performed within one week, with one cations [46,47]. One fish and one casein diet were left male per three females. The males were rotated after unspiked for control, while PCB153 was added in high the first oestrus cycle in case of infertility in the males, or low concentrations to both fish and casein diets, pro- which has been observed previously (data not shown). On GD16 females were separated into single cages ducing a total of six diets: Casein Control, Casein Low PCB and Casein High PCB, Fish Control, Fish Low PCB (Eurostandard Type III H) in order to monitor each lit- and Fish High PCB. The spiked low dose diet aimed at ter separately. Cages were checked twice daily for litters a PCB153 concentration similar to the concentration from GD18. The day of birth was denoted PND0. Pups typically found in farmed Atlantic salmon from Norway were weighed and handled every third day from PND5 [48], where PCB153 is the most prevalent congener. The until PND18 (Figure 1, Table 1). exposure to PCB153 from the diets with the spiked high Most animals were sampled on PND19. For the high concentrations represented a worst case scenario with level PCB groups and the Fish Control group, one male repeated consumption of fish with extremely high levels and one female sibling per litter were weaned on of PCB153. The intake of food was monitored by weigh- PND21, and monitored until week 16. All pups from ing any uneaten food daily, and calculation of the dose low dose groups were sacrificed on PND19 due to a low per kg body weight (BW) was done for gestation and lactation. Calculated doses ingested by the dams in the high dose groups were similar to the doses previously shown to produce persistent changes in spontaneous behaviour after single exposure on Postnatal day (PND) Mating 10 [13]. Dams with 30 g body weight consuming a max- 1 week 1 week 2 weeks ~3 weeks week 4-16 Monitoring Acclimation Gestation Behavioural testing imum of 12 g feed with 1500 ngCB153/g feed during lactation would have a peak daily intake of PCB153 of 1.7 μmol/kgBW*day. Feed and thus contaminant intake Experimental feed with PCB153 Clean chow diet by the dams peaked around PND11, which coincides Figure 1 Experimental outline. The experiment was performed with the BGS in mice [8]. The estimated exposure of during 16 weeks. Mice offspring were treated and handled as each pup would be highly influenced by pup body indicated. GD = Gestation day, PND = Postnatal day. weight and number of pups per litter. The lactational Weaning: PND21 Arrival GD0 Birth: GD21/PND0 PND 5 PND 8 PND 11 PND 15 PND 18 week 16 Behavioural testing and sampling Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 4 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 Table 1 Observations of physical development and behavioural tests in murine offspring Physical markers of development PND5 PND8 PND11 PND15 PND18 Week 16 Weight ++ + + + + Freeing of pinnae ++ Fur development ++ Incisor eruption ++ + Eye opening ++ Behavioural testing PND5 PND8 PND11 PND15 PND18 Week 16 Grasping reflex ++ Righting reflex ++ Climb/hang test ++ + Auditory startle ++ + Visual placing + Cliff drop aversion + Forelimb strength ++ Open field ++ Startle reflex (ASR/PPI) + Behavioural tests were performed on all pups in a litter from postnatal day (PND) 5 to PND18 and at 16 weeks of age. +: Signifies that the test was performed that day. number of litters in the Casein Low PCB group. All ani- produces a sharp sound without visible movement. mals were marked for identification and tracking of Except for hang/climb all reflex tests were scored 0-1, developmental history. Pups were given water and stan- where “1” is the display of characteristic behaviour [43]. dard rodent diet in pellets (chow, 10% fat) ad libitum For the hang/climb-test, graded levels of hanging and after weaning. Providing clean chow during development climbing were scored. Climbing was defined as moving and successfully replacing at least one fore- and one would imply that any observable toxic effects must have been caused by the exposure during early development. hind limb. The recordings were scored such that: 0 = no Pups were weighed weekly and inspected daily from hang, 1 = 1-14 sec, 2 = 15-29 sec, 3 = 30-44 sec, 4 = 45- Monday to Friday. Aggressive males were separated into 59 sec, 5 = > 60 sec, 6 = successfully climbing, replacing individual cages. All males were separated one week two limbs, 7 = successfully replacing three limbs, and 8 before behavioural testing in week 16, to minimize the = successfully replacing all four limbs while climbing. differences in caging conditions and stress level before Additionally, pups were tested for visual placing and the final behavioural test. cliff aversion [7] after eye opening on PND15. Forelimb strength was measured for pups on PND15 and -18 by a Behavioural testing grip strength meter (San Diego Instruments Inc., San From PND5 to PND18 the pups were observed and Diego, USA), and on PND18 pups were tested for anxi- tested for physical markers of development, reflexes and ety and spontaneous behaviour in an open field. The motor-function (Table 1). Behavioural tests were done open field arena was a (42 × 42) cm black polyethylene at the same time of day for each age-group, in a dedi- arena with 25 cm high walls, enclosed by white sheets cated room with the same temperature and lighting as hanging from the ceiling, and lit by indirect lighting to the housing conditions. All pups in a litter were tested create a uniform environment and exclude visual cues. by a single experimenter who was blinded to the treat- For the data analysis the open-field arena was divided ment. Where possible, at least one pup was left in the into two zones, a periphery, defined as a 7 cm wide area cage at any time during testing to relieve maternal along the walls, and a centre zone defined as the (28 × stress. On PND5, -8 and -11 all pups were tested for 28) cm square in the centre. On PND18 the animal was behavioural reflexes, based on the Fox battery [7] with placed in the centre of the field and latency to leave the centre was measured. In week 16 the animals were modifications previously described [63]. The individual tests were chosen based on experience from previous placed in an adjacent chamber, allowed to acclimate for trials [43,64]. The following tests were performed as two minutes, before a door to the open field was opened described by Folven et al. [43]: righting, hind limb and latency to enter the open field was measured. On grasp, a combined hang/climb-test and auditory startle PND18 movement was recorded for three minutes and reflex, with a small modification for the startle reflex in week 16 for 10 minutes, using a digital camera and test which was tested using a metal “click-box” that the behavioural tracking software SMART (San Diego Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 5 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 Instruments Inc., USA). The tracking recorded the intensities has previously been shown to be from 40-60% latency to leave the start zone (PND18 only), latency to [66]. Based on suspected gender differences in the PPI enter the field (week16 only), total distance travelled, response a decision was made apriori to separate time spent resting, the number of entries into the cen- genders. tre, and the total time spent in centre and periphery, Shortly before the final sampling two males from respectively. Also recorded was the distance travelled Casein High PCB and one male from Casein Control along the perimeter (% of total distance), and speed of were scanned with Magnetic Resonance Imaging (MRI) as a preliminary check for macro-anatomical changes in movement in the perimeter. The arena was wiped with the brain. No apparent changes in ventricle size or gross 70% ethanol and water between trials. The test order was randomized with respect to treatment group, litter brain morphology following the repeated perinatal PCB- and individuals in the litter. The test was performed at a exposure were seen, and the remaining animals were consistent time of day. not scanned. Complex behaviours: prepulse inhibition Sampling The startle response is an unconditioned response to an At PND19 most pups were euthanized and the liver was audible, tactile or sensory stimulus. The response is plas- sampled and frozen at -20°C until PCB analysis. At 16 tic, in that it can be modulated depending on other exter- weeks of age, remaining animals were euthanized by nal cues and repeated stimuli. Prepulse inhibition (PPI) is exsanguinations. Blood was drawn from the heart with a a phenomenon where a weaker stimulus prior to the syringe and mixed with 10 μl heparin (2.02 units/μl), main stimulus attenuates the response to a subsequent immediately centrifuged at 4°C, 2500 rpm for 5 minutes startle response. This modulation of the startle response and the serum frozen at -80°C until biochemical analyses is highly conserved in mammals, and has been used and (within two months). Liver enzyme analyses were per- discussed as a cross-species measure of sensorimotor gat- formed on serum samples to assess liver damage. Blood ing [28,65]. Abnormalities in PPI have been linked to sugar levels and blood lipids were also analysed in order neuropsychiatric disorders and diseases like schizophre- to examine differences related to early toxic exposure nia, Alzheimer’sand Huntington’s disease [28,65]. At 16 with different maternal diets. Analyses were performed weeks of age, the animals were tested for acoustic startle using a MAXMAT PL multipurpose diagnostic analyzer response (ASR) with prepulse inhibition (PPI) using the system (MAXMAT S.A. Montpellier, France). The fol- “Startle box” (Med Associates Inc., USA). The mice were lowing kits from DIALAB GmbH: Alanine transferase/ placed in a grid floor animal holder in the sound-attenu- Glutamate Pyruvate Transaminase (ALAT) with Pyri- ating cubicle, acclimated for five minutes with 60 decibel doxal 5’ phosphate, Alkaline Phosphatase (ALP), total (dB) ambient white noise and thereafter exposed to 40 Cholesterol (Chol), Gamma-glutamyl transferase (GGT) acoustic stimuli (Additional file 3). All stimuli were pre- and High Density Lipoprotein cholesterol (HDL); and sented as 120 dB white noise signals over a 60 dB ambi- from MAXMAT: Glucose (Glc), Lactate Dehydrogenase ent white noise. Prepulses (73, 75, 80 or 85 dB) were (LAD), and Triglycerides (TG) were used for serum ana- presented in pseudorandom order 100 ms prior to the lyses. Analyses of ALAT and GGT failed in a number of 120 dB main stimulus (Additional file 3). Inter-trial inter- samples and were omitted from results. Two additional vals varied randomly from 10 to 20 seconds. The ASR samples were omitted from the analysis due to outlying was defined as the peak amplitude after the 120 dB main results caused by technical problems during sampling. stimulus, subtracted the peak amplitude caused by spon- Additionally, serum was analysed for free Triiodothyr- taneous activity for 200 ms prior to prepulse presenta- onine (fT3), free Thyrosine (fT4) by RIA and Thyroid tion. Mean PPI for each prepulse intensity was calculated Stimulating Hormone (TSH) by IRMA, according to the as percent reduction in mean startle response with pre- manufacturer’s specifications (DRG international, Inc). pulse (ASR-pp) compared to the startle response without Only one replicate was analysed per sample due to lim- prepulse (ASR-simple, Additional file 3) according to the ited amounts of serum. formula (ASR-pp/ASR-simple)*100-100.Habituation was Concentrations of PCB153 in pup livers on PND19 calculated from the differences in mean response of the and abdominal fat in week 16 was analysed by Gas five last ASR-simple compared to the first five ASR-sim- Chromatography/Mass Spectrometry (GC/MS) in SIM ple, according to the formula [(ASR block3-ASR block1)/ mode, based on the same methods as used to analyse ASR block1]*100-100. With these approaches, normal the feeds [46,47]. inhibition of the startle response and habituation produce high negative values, whereas lack of inhibition and habi- Statistical analyses tuation gives values close to zero, or positive values. PPI The statistical methods employed here keep the litter as in untreated male BALB/c with this range of prepulse the experimental and statistical unit. This is highly Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 6 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 recommended when working with multiparous species, analyses and for analysis of PPI. The non-parametric as pups from the same litter are more similar than pups Kruskal-Wallis test of independent samples, followed by from different litters [67,68]. Mann-Whitney U pair wise comparison were used for comparison of means. Due to small sample sizes the Statistical models “Exact significance-test” was used. Significance levels All analyses aimed to assess how the response character- were set at alpha = 0.05 for all analyses. Because of missing data, two litters were excluded istics were influenced by the independent factors “diet” from the analysis of eye-opening: One litter in Fish High (fish or casein), “PCB153 level” (control,low,high),and PCB with 7 pups on PND12 and one litter in Casein “litter size” (1-10 pups) and their interactions. Inclusion of the dam as a random factor kept the litter as the sta- High PCB with 7 pups on PND13. Two litters in Fish tistical unit, as recommended [67,68]]A Linear Mixed Low PCB with only one pup were excluded from com- Effect Model (LME) [69]was employed to model influ- parisons of feed intake. Females were excluded from the ences on body weight, weight gain week 4-16 (corrected Open Field analyses in week16 due to technical difficul- for autocorrelation), Hepatosomatic Index (HSI = Liver ties performing the test. Males that died before week 16 weight*100/body weight),forelimbgripstrength,total were excluded from comparisons of body weight from distance travelled in the field, latency to leave the start week 4-16. zone (PND18) and latency to enter the field (week 16 only). The LME procedure accounts for the pups being Results grouped by the dam. For the reflex responses (PND5- Comparability among groups and litters 18) a generalized linear model (GLM) with quasi-/bino- There were no consistent, exposure related group differ- mial distribution was employed. The GLM model ences in body weight or mean feed intake among dams applied the response from each pup as a trial for the (Table 2). Reproductive success and litter size was not dam. In this way information about the number of pups different among groups that produced viable litters. The tested (trials) and the number of successful scores in the Casein Control group did not produce any viable litters. litter was included. Inclusion of the litter size as an The cause for the reproductive failure is not know, but independent variable provided a statistical test for the may reflect possible external stressors the first few days significance of the litter effect [68]. Additionally, in this after birth, which by chance affected this group the model the dam was included as a random factor repre- most. Several viable litters were born in other treatment senting the litter, due to differences in maternal care. groups on the same day. All diets were found to contain For the Open Field and the hang/climb test, scores were 10% fat and 17% protein. The vitamin supplements used measured as percent of maximum and compared using were identical for all feeds. Analysed concentrations of a variant of this model (GLMM), similar to the LME PCB153 in feeds are given in Table 2. Feed intake by procedure, but allowing for binomially distributed dams increased from approximately3.5 g/dayduring responses [70]. All models adjusted for uneven variance early gestation to a maximum of approximately 12 g/day of the residuals (heteroscedascity). The model selections during lactation. The feed intake by dams during lacta- were obtained from backward elimination. Due to the tion was strongly correlated with litter size (p < 0.001; reproductive failure in the Casein Control group, a full Figure 2). The feed intake during lactation and hence analysis including all possible terms was not possible. the calculated intake of PCB was significantly higher for The biological signal was then assessed through two Fish High PCB than for Casein High PCB (Table 2). separate models instead; model I used the influence from and interaction between litter size, diet and two Analyses of PCB153 levels PCB (low or high) only, disregarding the control Concentrations of PCB153 in pup livers at PND19 groups due to the missing Casein Control. Model II reflected maternal dietary intake. Pup liver PCB153 con- investigated the two-way interaction of litter size and centrations (Mean ± SEM) were 2.1 ± 0.5 ng/g, 9.6 ± PCB153 level among the fish groups (Fish Control, Fish 0.4 ng/g, 15.6 ± 5.7 ng/g, 2200 ± 300 ng/g and 1800 ± Low PCB and Fish High PCB). Where comparison of 200 ng/g ww for Fish Control, Casein Low PCB, Fish single groups was warranted, Tukey HSD Post hoc test Low PCB, Casein High PCB and Fish High PCB was used. Pearson’s product-moment correlation test respectively. was used to test the correlation between body weight and litter size in pups. Growth and development The above statistical analyses were performed by the Pup body weight was monitored from PND5-18 (Figure 3). statistical software R [71]. SPSS 15.0 for windows (SPSS Body weight of males and females are reported separately Inc., Chicago, IL, USA) was used for the nonparametric from week4 to week16 (Figure 4). The Fish Control comparisons of reproductive success, biochemical group generally had a higher body weight than pups in Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 7 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 Table 2 Feed concentration, body weight, reproductive output and feed intake by reproducing dams fed PCB153 in a fish or a casein-based diet Diet PCB153 Initial BW Final BW Repr. Litter size Intake gestation Intake lactation Gestation dose Lactation dose (n) ng/g feed dams dams succ % (g/day) (g/day) (μg/kgBW*day) (μg/kgBW*day) Casein Control 0.25 ± 0.12 n.d n.d 0 n.d n.d n.d n.d n.d (0) Fish Control 0.50 ± 0.07 28.9 ± 0.5 30.4 ± 0.2 33 4.7(4-5) 3.6 ± 0.2 7.7 ± 0.4 0.05 ± 0.00 0.12 ± 0.00 (3) Casein Low PCB 6.53 ± 0.12 29.1 ± 0.5 31.1 ± 0.9 33 6.3 (5-9) 3.6 ± 0.1 8.3 ± 0.7 0.73 ± 0.06 1.72 ± 0.23 (3) Fish Low PCB 6.57 ± 0.67 29.6 ± 0.8 31.3 ± 0.6 78 7.0 (4-9) 3.6 ± 0.1 8.6 ± 0.5 0.69 ± 0.03 1.63 ± 0.11 (5) (7) (7) Casein High PCB 1400 ± 60 28.4 ± 0.9 31.3 ± 1.5 56 5.8 (4-8) 3.9 ± 0.2 8.1 ± 0.3* 170 ± 10 350 ± 20* (6) Fish High PCB 1500 ± 300 30.0 ± 1.0 29.6 ± 0.7 56 6.8 (4-10) 3.5 ± 0.2 9.2 ± 0.3* 160 ± 10 420 ± 20* (5) Data are presented as mean ± SEM. Number of litters per group in brackets. Four litters were born in the Casein Control group, but no pups lived until postnatal day 5. Initial BW = BW after acclimation, final BW = BW end of weaning. Repr. succ: = percent successful reproduction in relation to the number of mated females. Litter size is given as mean (min-max). Feed intake during gestation (GD0-21) and lactation (PND0-21) was based on daily monitoring of feed intake. Daily doses were calculated based on average feed intake in grams and PCB153 concentrations in feeds, rounded off to the nearest ten ng/g for the high levels. Two females with single pups in Fish Low PCB were excluded from the calculation of litter size, feed intake and dose during gestation and lactation, but were included for comparisons of initial and final body weight (BW) of reproducing dams. n.d: No data due to reproductive failure. *: Groups that share a symbol are significantly different (Student’s t-test: p < 0.05). the PCB exposed groups. This trend persisted from lacta- male and female body weight until adulthood (p < 0.05 tion until week16, but was significant only in week 16. and p < 0.001) respectively. The mean litter size of the Fish Control group was slightly less than the Casein High PCB and Fish High Developmental effects of litter size, maternal diet and PCB groups (Table 2). The pup weight gain during lacta- PCB exposure tion was mainly influenced by litter size (Figure 2) and In the present study, the litter size was a prominent and was not significantly affected by the protein source (fish recurring factor which affected the behaviour and devel- or casein) or the PCB level. opment in a predictable and consistent manner. Physical Females in the Fish Control group were significantly heavier than females in Casein High PCB (p < 0.01) at week16 after correction for litter size (Figure 4, left Fish Fish Control Control panel). LME (adjusted for repeated measurements) Fish Fish Low Low demonstrated that the initial litter size had an effect on 12.5 Fish Fish High High Casein Casein Low Low Casein Casein High High 16 10.0 Intake Lactation Intake = 0.69** R Gestation 7.5 R 5.0 = 0.51** R = 0.01 2.5 0246 8 10 12 05 10 Litter size (count) Litter size (count) 5 5 8 8 11 11 15 15 18 18 Figure 2 influence of litter size on pup body weight and Postnatal day maternal feed intake. Left panel: Negative correlation was observed between pup bodyweight and litter size. Right panel: Figure 3 Body weight gain in offspring during lactation.Body Positive correlation was observed between maternal feed intake weight was measured on Postnatal day 5, -8, -11, -15 and -18. Each during lactation and litter size. Feed intake during gestation was not point with error bars represents the mean body weight ± SEM per affected by litter size. **: p < 0.001. group, in pups of both genders. Body weight (g) Pup bodyweight (g) Feed intake, dam (g) Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 8 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 At PND18 males spent more time in the centre of the Females Males 40 40 Fish Fish Control Control open field, spent less time moving fast and travelled a Fish Fish High High Casein Casein High High shorter distance than females (all groups compared, p < 30 30 0.001, p < 0.05 and p < 0.05 respectively). No other gender effects were seen. For the remaining analyses 20 20 genders were therefore grouped. On PND18 the fish groups left the central starting 10 10 zone significantly faster, and spent more time moving fast than casein groups (p ≤ 0.01 and p < 0.05, respec- tively). A trend towards lower resting time in the fish 0 0 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 14 14 15 15 16 16 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 14 14 15 15 16 16 groups than the casein groups was also seen (p < 0.08), Age (weeks) Age (weeks) indicating higher reactivity in the fish-groups regardless Figure 4 Body weight gain in juvenile mice week 4 to 16.Each of PCB exposure. No differences were observed for total point with error bars represents the mean body weight ± SEM in distance travelled; time spent moving slow and fast, female (left panel) and male (right panel) BALB/c mice exposed to PCB153 during gestation and lactation. The data include animals time spent resting or permanence time in the centre or that survived until week16. the periphery. Open field in week 16 Similar toPND18,inweek 16 malesfromthe fish development, such as body weight, fur development on groups entered the open field significantly faster than PND5, incisor eruption on PND11 and eye opening on PND14 were negatively related to litter size, with con- the casein group (p < 0.01). No effects of PCB exposure sistent and significant effects or trends in the two dif- were seen on the latency to enter the field or other ferent factorial models (data not shown). Regarding parameters tested. reflex development, a higher litter size significantly reduced the success rate in the hind limb grasp test Acoustic startle response with prepulse inhibition in (PND5), the hang/climb test (all days) and the startle week16 reflex (PND11) in both factorial models (p < 0.05) PPI tests showed large variability within the groups in Results for the forelimb grip strength were ambiguous. both genders. The small sample size and large variance Statistical analyses by model I showed that a maternal precludes statistical analyses, and the PPI test would fish diet was related to higher body weights in pups on need to be repeated with larger groups for conclusive all days from PND5 to PND18 (p < 0.05) after correc- results, and to determine possible diet, PCB exposure or litter effects. Although there were no significant differ- tion for the effect of litter size. The higher body weight ences among groups or genders, certain aspects of the may be the reason why a maternal fish diet was also findings deserve mentioning. This study shows that related to more fur on PND5 (p < 0.05), and tended to among the males, both of the PCB exposed groups give more pups with erupted incisors on PND11 (p < showed consistently less prepulse inhibition than the 0.1) as well as more pups with a startle response on controls (Additional file 4), although not significantly PND15 (p < 0.1). different. The reduction in startle response in exposed PCB exposure was not significantly related to body males was less than previously seen for BALB/c [66]. weight, growth or behavioural development. However, However exposed groups were not significantly different the timing of incisor eruption and eye opening appeared from the control group, and conclusions cannot be less synchronized in the PCB exposed pups than the drawn. controls. Comparing to Fish Control, the PCB153 exposed groups had earlier incisor eruption than the control group, but by PND11 the Fish Control was the Habituation to the acoustic startle response only group with full development of the trait. Similarly, A reduced startle response in the last set of simple sti- pups from Fish Low PCB and Fish High PCB had muli compared to the first set of simple stimuli indicates started eye-opening on PND12, whereas the pups from habituation, which is the expected response to repeated the Fish Control group had not. By PND14 there was stimuli. Noticeably, in males, both of the PCB exposed no difference among groups. groups showed the opposite response to habituation (Figure 5). The mean startle response was in fact higher Spontaneous behaviour in the open field PND18 in the last block of stimuli than at the start, indicating The open field used for assessment of spontaneous increased fear in that group, and no adaptation to the behaviour, anxiety and reactivity at PND18, showed situation. The large individual variability and the small differences in spontaneous behaviour among genders. dataset render conclusions difficult. Bo Bod dy y w we eiig ght ht ( (g g) ) Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 9 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 Table 3 Group size, body weight and hepatosomatic Females Males index for male and female offspring Fish Control Gender Group N BW BW HSI PND21 Week16 Week16 Fish High female Fish Control 3 16.9 ± 0.5 29.9 ± 1.1 4.97 ± 0.14 Casein High 6 13.0 ± 1.1 25.5 ± 0.8 4.36 ± 0.10 PCB Casein High ab Fish High PCB 5 14.9 ± 1.8 25.9 ± 1.3 4.65 ± 0.20 -4 -40 0 -2 -20 0 0 0 20 20 40 40 -6 -60 0 -4 -40 0 -2 -20 0 0 0 20 20 40 40 60 60 male Fish Control 3 16.6 ± 1.0 33.0 ± 2.3 4.38 ± 0.20 Habituation (%) Casein High 4 14.0 ± 1.2 30.6 ± 1.1 4.87 ± 0.38 Figure 5 Habituation to the acoustic startle stimuli in week 16. PCB Reduction in startle response at the end compared to at the start Fish High PCB 4 17.6 ± 3.1 27.0 ± 1.8 4.65 ± 0.62 of the trial. The bars and the error bars represent the percent The animals were exposed to PCB153 throughout gestation and lactation, via habituation (mean ± SEM). A negative value indicates a normal maternal consumption of spiked feeds based on fish or casein. High dose habituation response, with a lower startle response at the end than feeds (Fish High PCB and Casein High PCB) contained ~1500 μgPCB153/kg at the start of the trial. Sample sizes were (females/males): Fish feed. Data are presented as mean ± SEM of offspring that survived to week16. Control: n = 3/3; Fish High: n = 4/5; Casein high, n = 6/4. HSI = Hepatosomatic index, BW = body weight, PND = Postnatal day. Groups with different superscript letters are significantly different, within the same gender (Mann-Whitney U, p < 0.01). General observations of behaviour in PCB exposed males In week 4-16 allfemalegroupsand themalecontrols Casein High PCB group only (p < 0.05). TSH was below had 100% survival. Mortality was only observed in PCB the level of quantification (0.2 pg/L) for all samples, so exposed males. By week 12 the males in the Casein comparisons could not be made. High PCB group had a survival of 67% (4/6) and by week 13 males in the Fish High PCB group had a survi- Discussion val of 83% (4/5; Additional file 5). No deaths were The study aimed to elucidate if a worst case scenario caused by male aggression. However, all males in the with high maternal PCB153 intake through fish con- Casein High PCB group were separated into single cages sumption during brain development can be expected to since they developed aggressive behaviour. give any observable behavioural or developmental effects. Maternal nutrition is important for offspring Biochemical analyses of PCB concentrations, blood lipids development, and fish is also a known source of many and liver enzymes week 16 beneficial nutrients. The possible ameliorating effect of a PCB153 was analysed in abdominal fatin week16to maternal high fish diet was hence compared to a similar elucidate if maternal diet during gestation and lactation intake of PCB153 in a diet without fish. had an effect on pup metabolism and lasting body bur- Early pup development and behaviour as well as adult dens of PCB153. After 12 weeks on a standard chow behaviour and health were assessed as toxic effects have diet, groups still had PCB153 levels that reflected the previously been shown to manifest with increasing age maternal PCB intake during gestation and lactation. [10-12]. PCB153 concentrations were, Fish Control: 11.4 ± 2.7 The observations of this study suggested that continu- ng/g ww, Casein High PCB: 7000 ± 600 ng/g ww and ous maternal exposure to the single PCB153 congener Fish High PCB: 7000 ± 700 ng/g ww (Mean ± SEM, during gestation and lactation had little effect on para- high level groups rounded off to the nearest 100 ng/g meters of physical and neurobehavioural development in ww). Serum contents of blood lipids, liver enzymes and the offspring, but that survival and behaviour in early glucose showed minor differences only in levels of TG adulthood might have been compromised. The study among females (Additional file 6). There were no differ- suggested that PCB153 exposure might have affected the ences in blood lipids, liver enzymes or glucose among synchronization of physical development, such as eye the males. For the females in week 16, the Fish Control opening and incisor eruption. This tendency to dis- group had the highest HSI (Table 3), which coincided turbed synchronization of development has previously with a higher body weight of females in the Fish Control been observed in rats [72]. Disturbances in teeth devel- group (Figure 3). opment has also been observed in children exposed to PCBs during the so-called Yusho disasters where PCB Thyroid status contaminated rice-bran oil was ingested [73,74]. Nutri- No differences in fT3, fT4 or the ratio fT4/fT3 were tional effects on pup development were also present as found among groups (Figure 6). Comparing the genders, trends; as pups of dams fed the fish diets had an accel- males generally had lower fT3 and higher ratio fT4/fT3 erated development of physical traits, increased auditory than females, which was statistically significant in the startle reflex, and were more active in the open field Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 10 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 the mortality of the juvenile offspring in males. How- ever, the biochemical and behavioural evidence of last- 20 20 ing adverse effects of perinatal PCB153 exposure in adult mice were scant, possibly as a cause of the low ** sample size. The biochemical and behavioural data from week 16 are thus inconclusive. 15 15 Nutritional aspects Considering the nutritional aspect of the study, all diets had the same levels of nutrients and fulfilled require- ments for vitamins, minerals, protein and lipids (Addi- 10 10 tional file 7). An effect of nutrition on physical female female development has previously been seen with selenium male male supplementation, which increased fur development in neonate mouse pups [43]. It is possible that the more 6 6 nutritionally complex fish diets, which contained both fish, casein, fish oil and soy-oil, provided a more optimal nutrient composition which was beneficial during early 5 5 development. However, without a demonstrable adverse effect of PCB153 any possible ameliorating effects of fish could not be ascertained. 4 4 Neurobehavioural tests Previous studies have documented the validity and effi- 3 3 ciency of the neurobehavioural tests used, and have detected neurobehavioural differences in mouse pups using a similar test battery [43,64]. The fact that subtle 2 2 changes related to litter size were consistently detected in a predictable manner underlines the sensitivity and utility of the statistical methods. The effect of litter size, 0.4 0.4 when present, always affected the results in the expected direction, i.e. larger litters produced smaller and less agile pups. This again supports that the statistical meth- ** 0.3 0.3 ods employed would detect subtle changes where these were in fact present. Thus, any effects of PCB153 larger than the effect of litter size would presumably be detected. 0.2 0.2 Animal model Interpretations of the open field data at weaning were ambiguous, and the results did not provide a good indi- 0.1 0.1 cation of effects on anxiety from diet or dose PCB. The Fish Control Fish High Casein High interpretation of open field data must be performed Figure 6 Thyroid hormones in serum at week16 in murine with care [75]. The ambiguous results could be influ- offspring. Hormone concentrations of free triiodothyronine (fT3, enced by the immature exploratory behaviour in pre- upper panel), free thyroxine (fT4, middle panel) and the ratio fT4/fT3 weanling mice [64], but may also reflect the nature of (lower panel) in pups exposed to PCB153 via gestation and lactation. The boxes show the median and the inter-quartile range. BALB/c mice used. BALB/c mice are phenotypically * indicates significant difference between genders (Mann-Whitney U neophobic [76], which may have reduced the degree of pair wise comparison, p < 0.05). exploration and range of behaviour displayed in the open field, thus limiting the possibilities to discern compared to casein fed pups. Neither diet nor PCB effects of exposure among groups. Selection of a model exposure greatly affected development until adulthood, with higher reactivity to the test, such as the less but the maternal fish diet may have caused increased anxious C57BL/6 might have improved the ability to body weight in pups. PCB exposure may have increased discern effects among groups. Fr Fre e e e T4 T4 p pm m o oll//ll fr fre ee eT T 4 4 /fr /fre ee e T T 3 3 Fr Fre e e eT3 T3 p pm m o oll//L L Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 11 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 Statistical methods were exposed to sufficiently high levels during the vul- Above all, this study highlights the importance of taking nerable phase to potentially produce effects. Analyses of litter size into consideration in statistical evaluations of tissue levels in pups showed that they had high levels of neurodevelopmental responses. The findings support PCB153 in liver and fat on PND19, indicating a high previous observations showing that the litter effect may exposure to this di-ortho PCB congener throughout the be large and should always be included as a confounding period of BGS. The concentrations of PCB in the brains factor or predictor of the responses [7,67,68]. Not taking of pups which showed aberrant behaviour in previous into account the effects of intra-litter correlation and the studies [17] were low at the time of behavioural testing. litter size, may give an erroneous impression of group The current results suggest, however, that the effects are differences that are in reality caused by differences in lit- greater with exposure to a single, high bolus dose ter size and maternal care [68]. Particularly in studies of around BGS than with continuous exposure or slow teratogenic effects, or after maternal exposure of pups, accumulation throughout gestation and lactation. the litter effect may be prominent. It is essential to keep Neurotoxicity of the di-ortho PCBs have been shown in mind that the rearing condition (litter size) and the to manifest with age [10,12,13], but in the present study, toxic exposure (dose) of each litter are mutually depen- no significant effects in cognitive function or sensorimo- dent and are defined by the maternal care provided by tor gating were seen. However, the trends to difference each individual dam. Including both litter size and dam in PPI, the observed increase in male aggression in the analyses is therefore essential to discriminate throughout the trial and a corresponding lack of habi- between the individual effects, and will also provide a sta- tuationinthe ASRtestof the PCB exposed groups, tistical measure on the magnitude of the litter effect. indicate an increased stress level or increased anxiety in If any effects of PCB153 were indeed present and of males. The lack of habituation in the ASR-test may also the same magnitude as the effect of litter size it would be interpreted as a lack of ability to comprehend and have been detected, even with sample sizes as low as adapt to the novel situation which the startle-box repre- three animals. The benefit of the factorial design is that sents. The study of ASR/PPI needs to be repeated with it artificially increases the sample size when groups are a larger sample size for conclusions regarding the obser- combined for investigation of the factor they have in vations of lower PPI in PCB153 exposed males. common (diet, exposure level or litter size). This means that even though sample size is low for some groups, Biochemical effects of PCB153 the total number of litters exposed to i.e. “high level” is In previous epidemiologic studies of thyroid hormone dis- the sum of Fish High PCB and Casein High PCB ruption, exposure to the background levels of persistent together, which is 5+6 = 11 litters. Thus the statistical organic pollutants (POPs) in Scandinavia had little effect power for detecting effects of a high dose PCB153 is on thyroid hormones in breast-fed infants [77]. In con- increased compared to using a one-way ANOVA where trast, severe thyroid hormone disruption and impacts on the groups are investigated separately. For the factor cognitive function were seen after consumption of highly “litter size” it means investigating all litters with the PCB contaminated rice-oil [73], potentially containing same number of pups, regardless of diet or dose, to see traces of furanes and unidentified contaminants as well as if they are more similar than litters of different sizes. PCB. A combination of mixture-effects and exposure The low sample size however obstructs detection of dif- levels is likely to be of importance to the thyroid disrupt- ferences among groups and the potentially ameliorating ing effects seen in other studies, whereas the PCB153 con- effects of maternal nutrition. Although reproductive out- gener alone at the present doses did not affect the thyroid put in this study was lower than expected, the differ- hormone levels or the blood parameters investigated. Only ences in reproduction appeared not to be related to a slight difference in TG levels was seen among females. PCB153 exposure. However, the TG levels were seemingly reduced by PCB exposure, which is in contrast to previous findings [78] Behavioural and cognitive effects of PCB153 where exposure to a mixture of POPs increased the levels It seems to be a recurring finding that organohalogen of TG in rats. Since the TG levels in males showed no exposure at low doses that do not affect growth or phy- consistent differences related to PCB153 exposure, the sical development, still produce aberrant behaviour at higher TG and also HSI in Fish Control females may be later stages [11-13,17,59]. As concerns the present expo- explained by the higher body weight sure level, the repeated doses to the dams were in the range of the single exposure doses (1.4 μmol/kgBW*day) Human relevance of the study regarding neonatal PCB that have been shown to produce aberrant behavioural exposure effects after a single oral dose on PND10 [12,13,56]. The findings imply that resource availability, nutrition, Thus it was assumed that pups in the present study maternal care and social environment during gestation Haave et al. Behavioral and Brain Functions 2011, 7:3 Page 12 of 14 http://www.behavioralandbrainfunctions.com/content/7/1/3 and early development affect development and beha- Additional file 7: Concentrations of nutrients in the experimental viour to a much larger extent than the present exposure feeds. Table of analysed concentrations of protein, fat and selected vitamins and fatty acids in the casein- and fish-based diets. to PCB153. In mice these parameters are defined by the litter size. This suggests that the developmental starting point may outweigh other influences. The importance of rearing conditions and nutrient on development and Acknowledgements The authors wish to express their gratitude to Aase Heltveit, Lars-Erik behaviour has recently been reviewed [58]. The impor- Pindard, Edel Erdal and Jacob Wessel at NIFES for technical assistance. tance of maternal care and nutrition also supports Thanks to Dr. Frits A. Thorsen at the Institute of Biomedicine, University of recent studies in children [37], where Docosahexaenoic Bergen, for performing the MRI, Gaute Velle at the University of Bergen for editing figures, and all other personnel at NIFES involved with feed- and Acid (DHA) and selenium, as well as breastfeeding were tissue analyses. found to protect against adverse effects of environmen- This work was funded by the EU-project “AQUAMAX"; EU-Contract no. tal exposure in early childhood [37]. Breastfeeding and 016249-2, and the National Institute of Nutrition and Seafood Research (NIFES). maternal care have also been suggested to modify the effects of contaminants in human behavioural develop- Author details ment [79]. National Institute of Nutrition and Seafood Research (NIFES), P.O. box 2029 Nordnes, N-5817, Bergen, Norway. Department of Biology, University of Bergen, P.O. box 7803, N-5020, Bergen, Norway. Department of Biological Conclusions and Medical Psychology, University of Bergen, Jonas Lies vei 91, N-5009, Exposure of mice offspring to the single congener Bergen, Norway. The Norwegian Forest and Landscape Institute, Fanaflaten 4, N-5244 Fana, Norway. PCB153 via the dam did not result in effects on physical development and behaviour that exceeded the biological Authors’ contributions influence of litter size. Synchronization of development MHA took active part in designing the project, preparing the experimental diets, animal housekeeping, collecting behavioural data, tissue sampling; such as eye opening and incisor eruption may have been performing the statistical analyses, writing the original draft and completing compromised by neonatal PCB exposure. A maternal the manuscript. ABE took active part in preparation of the experimental fish diet increased pup body weight and accelerated phy- diets, animal housekeeping, data collection and statistical analyses and performing the behavioural tests. FJE was involved in selection and design sical development, but did not alter pup reflex develop- of the ASR-PPI and the analysis of ASR-PPI-data. EHE selected, implemented ment or behaviour to a great extent. Although a larger and validated the statistical models using the R-software. TBR and AKL took sample size would increase the statistical power and part in designing the study and in revising and proofreading the manuscript. All authors read and approved the final manuscript. would help detect differences, it was shown that random variables, such as litter size have a great impact on pup Competing interests development and behaviour. This needs to be consid- The authors declare that they have no competing interests. ered in experimental design and statistical analyses. The Received: 1 September 2010 Accepted: 13 January 2011 study demonstrated that subtle differences can be Published: 13 January 2011 detected using the current methods and statistical treat- ment of the data. References 1. Guvenius DM, Aronsson A, Ekman-Ordeberg G, Bergman A, Noren K: Human prenatal and postnatal exposure to polybrominated diphenyl Additional material ethers, polychlorinated biphenyls, polychlorobiphenylols, and pentachlorophenol. Environ Health Perspect 2003, 111:1235-1241. 2. Noren K, Meironyte D: Certain organochlorine and organobromine Additional file 1: Composition of experimental diets. Table of dietary contaminants in Swedish human milk in perspective of past 20-30 years. components in the fish and casein based diets, given as g/kg feed. Chemosphere 2000, 40:1111-1123. 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