Early Developmental Neurotoxicity of a PCB/Organochlorine Mixture in Rodents after Gestational and Lactational Exposure

Wayne J. Bowers*,1, Jamie S. Nakai*, Ih Chu*, Michael G. Wade*, David Moir*, Al Yagminas*, Santokh Gill{dagger}, Olga Pulido{dagger} and Rudi Meuller{dagger}

* Systemic Toxicology and Pharmacokinetics Section, Healthy Environments and Consumer Safety Branch, and {dagger} Toxicology Research Division, Health Products and Foods Branch, Health Canada, Ottawa, Ontario K1A 0L2, Canada

Received July 9, 2003; accepted September 12, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The developmental and neurobehavioral effects of gestational and lactational exposure to a mixture of 14 polychlorinated biphenyls (PCBs) and 11 organochlorine pesticides was examined and compared against the commercial PCB mixture Aroclor 1254. The mixture was based on blood levels reported in Canadian populations living in the Great Lakes/St. Lawrence basin. Pregnant Sprague-Dawley rats were dosed orally with 0.013, 0.13, 1.3, or 13 mg/kg of the chemical mixture or 15 mg/kg of Aroclor 1254 from gestation day (GD) 1 to postnatal day (PND) 23. The highest mixture dose decreased maternal gestation and lactation body weight, and produced high mortality rates (80% overall) and reductions in offspring weight that persisted to adulthood. Aroclor 1254 produced smaller but persistent decreases in offspring weight without affecting maternal weight or offspring mortality. Aroclor 1254 and 13 mg/kg of the mixture produced comparable decreases in maternal and offspring serum T4 levels and comparable alterations to maternal thyroid morphology. Aroclor 1254 delayed the righting reflex and ear opening, accelerated eye opening, and reduced grip strength at PNDs 10–14. The mixture at 13 mg/kg delayed negative geotaxis in addition to delaying righting reflex and ear opening and reducing grip strength but had no effect on eye opening. Lower mixture doses (0.13 and 1.3 mg/kg) also delayed ear opening but affected no other parameters. Developmental exposure to the chemical mixture was found to be more toxic than exposure to Aroclor 1254 and produced a different profile of effects on early neurodevelopment. This PCB/organochlorine pesticide mixture affects mortality, growth, thyroid function, and neurobehavioral development in rodents.

Key Words: behavior; PCB; neurotoxicity; chemical mixture; organochlorine; thyroid; DDT; developmental toxicity; Aroclor 1254; Great Lakes; thyroid.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Epidemiological studies have demonstrated a relationship between perinatal exposure to persistent organic pollutants (POPs) and neurological and behavioral disturbances in infants and children (Jacobson and Jacobson, 1997Go; Koopman-Esseboom et al., 1996Go; Patandin et al., 1998Go). For example, infants of mothers consuming large amounts of contaminated Great Lakes fish exhibit disturbances such as decreased intellectual capacity, altered attention and memory processes, reduced birth size and decreased growth rate, altered psychomotor development, and delayed sexual development (Jacobson et al., 1990Go). In addition, exposure to ambient levels of POPs has been associated with altered physical growth, immune function, and thyroid hormone function in infants (Koopman-Esseboom et al., 1994Go). Studies in animals have confirmed that contaminants like polychlorinated biphenyls (PCBs), metals, and organochlorine pesticides can disrupt behavioral functioning. For instance, PCBs produce deficits in motor activity (Hany et al., 1999aGo), learning (Lilienthal and Winneke, 1991Go), memory and attention (Daly et al., 1989Go; Schantz et al., 1997Go), responsiveness to aversive stimuli (Daly et al., 1989Go), neuromuscular development (Bernhoft et al., 1994Go), and sensory function (Crofton and Rice, 1999Go; Goldey et al., 1995aGo). Thus both human and laboratory studies have demonstrated that exposure to environmental contaminants like PCBs may alter neurobehavioral function.

While epidemiological studies have, by necessity, evaluated the effects of mixtures of chemicals in humans, animal studies of the health effects of environmental pollutants typically assess single chemicals, simple mixtures (e.g., binary mixtures), or commercial mixtures (e.g., Aroclor mixtures). The limited data that are available from animal studies suggest that the toxicological effects produced by single chemicals or simple mixtures may not adequately estimate the toxicity produced by the chemical mixtures (Carpenter et al., 1998Go; Yang, 1998Go). For instance, recent studies have demonstrated that environmental chemicals found in humans can interact to produce toxicological effects that are not evident after exposure to single chemicals. Examples include PCBs and methylmercury (Bemis and Seegal, 1999Go) and PCBs and pesticides (Carr et al., 2002Go). Indeed, even the neurotoxicity of commercial mixtures of structurally similar chemicals like the PCB mixture Aroclor 1254 can vary considerably between production lots where PCB congener profiles vary (Kodavanti et al., 2001Go). Thus available evidence suggests that exposure to mixtures of environmental chemicals may produce health effects that differ from those associated with individual chemicals. Because there are limited animals studies that use chemical mixtures comparable to human exposure profiles, it is unclear that animal toxicology studies using single chemicals or simple mixtures provide adequate estimates of the toxicity of complex mixtures found in human tissues.

The current study was designed to evaluate the health risks of a complex chemical mixture based on the profile of contaminants found in human blood from populations living in the Canadian Great Lakes region. Relative blood concentrations in these populations were used as the basis to generate the chemical mixture. Because our primary interest was on the effects of developmental exposure to the mixture, rats were exposed to the chemical mixture throughout gestation and lactation. A separate group of animals was exposed to Aroclor 1254, a known neurotoxic agent, as a positive control treatment and to compare the toxicological impact of this frequently used PCB mixture against the complex mixture based on human blood contaminant profiles. Although we focused on evaluating neurotoxicological health effects, we also evaluated a variety of other organ systems. Of particular interest is thyroid function, because the thyroid hormone is essential for the normal growth and development of both central and peripheral nervous systems (Porterfield, 2000Go; Porterfield and Hendrich, 1993Go). Given that many of the chemicals included in the chemical mixture used here may affect thyroid function (e.g., PCBs, hexachlorobenzene, chlordanes, methylmercury) (Bondy et al., 2000Go; Brouwer et al., 1998Go; van Raaij et al., 1991Go), we measured biomarkers of thyroid function in dosed dams and offspring.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
The chemical mixture was derived from studies that measured contaminant levels in the blood of human fish-eaters living in the Canadian Great Lakes basin (Cole et al., 2002Go; Kearney et al., 1999Go; Kosatsky et al., 1999aGo; 1999bGo). The specific profile was generated from a weighted average of lipid-adjusted PCB and organochlorine concentrations found in tissues as reported in these studies. Organochlorine compounds and PCB congeners contributing more than 1.5% to the total mass were selected for inclusion in the final mixture. Two PCB congeners (PCB 126, 169) were measured at levels below the 1.5% inclusion criteria, but, because of their known high toxicity, they were added to the mixture at one-half of the detection limits reported in these studies. The contribution of each chemical to the total mass was calculated, and this percent contribution (by mass) defined the chemical mixture. Aldrin; p,p'-DDE, p,p'-DDT, dieldrin, heptachlor epoxide (isomer B), hexachlorobenzene (HCB), ß-hexachlorocyclohexane (HCH), mirex, cis-nonachlor, and trans-nonachlor were purchased from Sigma-Aldrich Canada Ltd (Oakville, ON, Canada). PCB congeners 74, 94, 99, 126, 156, 169, 170, 187, 194, and 200 (BZ 200/IUPAC 201) were purchased from AccuStandard Inc. (New Haven, CT). PCB congeners 118, 138, 153, and 180 were purchased from Radian International (Austin, TX). Oxychlordane was generously donated by Julie Fillion of the Pest Management Regulatory Agency (Ottawa, ON, Canada). All chemical purities were at least 99%.

The mixture was prepared by initially weighing the individual chemicals into clean vials and transferring them into small volumes of Mazola corn oil using preservative-free diethyl ether (Sigma-Aldrich Chemicals, Oakville, ON, Canada). The diethyl ether was then removed from the corn oil solutions using a Savant Automatic Environmental Speedvac (Model AES 2000) and loss of ether was confirmed by weighing the corn oil samples before adding ether and after processing in the Savant. The mixture was prepared at its final concentration (by mass) using clean corn oil. Because the chemical mixture precipitated out of the liquid upon standing, 5% (weight/weight [wt/wt]) diethyl ether was reintroduced to the PCB/OC mixture as a co-solvent to ensure that all compounds remained in solution. Specific dosing solutions were prepared from the stock solution by serial dilution with corn oil containing 5% (wt/wt) of diethyl ether. Aroclor 1254 (AccuStandard, New Haven, CT, lot number 124191) was prepared at 15 mg/ml in corn oil with 5% diethyl ether. The control dosing solution consisted of corn oil containing 5% (wt/wt) of diethyl ether. The mixture composition was verified by an independent laboratory (Wellington Laboratories, Guelph, ON, Canada) using high-resolution GC-MS analysis.

Animals.
All treatment procedures and housing conditions conformed to the Canadian Council on Animal Care guidelines and had been reviewed and approved by Health Canada’s Institutional Animal Care Committee prior to the start of the study. Ninety-three nulliparious female Sprague Dawley rats (200–230 g) and 38 male Sprague Dawley rats (315–350 g) served as subjects. The animals were obtained from Charles River Laboratories, St. Constant, Quebec, Canada.

Upon arrival, the females were housed two/cage and the males were housed individually. The animals were housed in polycarbonate hanging cages measuring 35 cm (L) x 30 (W) x 16.5 (H) with shaved wood bedding in rooms maintained at 22 ± 2°C and 50 ± 10% humidity. The study sample consisted of two cohorts that were bred separately but otherwise treated identically.

One day after arrival in the housing facilities, the animals were switched to a reverse light cycle (lights on at 20:00h, lights off at 08:00h) over a 12-day period (1 h/day) and were maintained on this reverse cycle thereafter. Food and water were available ad libitum. Seven days after arrival, permanent identification chips (Model IMI-1000, Bio Medic Data Systems, Seaford, DE) were implanted in the females under light isoflurane anesthesia. The females were weighed daily beginning 10 days before the start of breeding. After three weeks habituation, breeding was conducted by placing two females into a male’s cage and monitoring the females twice daily for vaginal sperm plugs. Once a vaginal plug was detected (denoted as GD 0), the female was removed from the male’s cage and housed individually in a cage with shaved wood bedding.

Doses of the chemical mixture were based on a pilot study in which pregnant Sprague-Dawley rats were orally dosed with 0.15, 1.5, and 15 mg/kg/day from GD 10 to 17. There were no indications that any mixture dose affected maternal weight gain, reproductive rates, litter size, pup growth, development, or mortality. Based on these pilot study results, the mixture doses selected were 0.013, 0.13, 1.3, and 13 mg/kg/day. The control group was dosed with untreated corn oil and a separate positive control group was dosed with 15-mg/kg/day Aroclor 1254. The dose groups were assigned on a pseudo-random manner based on the sequence of pregnancy (vaginal plug detection). All females were assigned to dose groups over the entire period of breeding, and dosing assignment was independent of the sequence of pregnancy.

Dosing.
The pregnant females were orally dosed by placing 1-µl/g body weight of the assigned dose on a Teddy Graham® cookie (approximately 2.0 g) (Nabisco Ltd., Toronto, ON, Canada) and placing the uncovered cookies in a fume hood overnight to allow the ether to evaporate. The dosed cookies were delivered to pregnant rats the next day. All dams had been pre-exposed to cookies dosed with 250 µl of clean corn oil for 5 days prior to breeding. The females readily consumed the dosed cookies, and there were no cases where the females rejected or did not completely consume the dosed cookies. The dams were dosed from GD 1 to weaning at PND 23.

Starting on GD 18, the dams were monitored three times daily for parturition at 07:00, 15:00, and 23:00h. The day of birth was denoted as PND 0. The pups were counted on PND 0 but not weighed or handled. The pups were sexed on PND 1, and the gender was confirmed on PNDs 2, 3, and 4. Total body weights for males and female pups in each litter were obtained on PNDs 1, 2, 3, and 4. Mortality and morbidity were monitored daily, and dead pups were removed immediately. The litters were culled to eight pups on PND 4 by randomly selecting four males and four females (where possible) from each litter. Where fostering was required to obtain litters of eight pups, the pups were fostered from the same dose group if possible. The fostered pups were excluded from all analyses. The pups were individually identified on PND 4 with footpad injections (Ketchum Animal Tattoo Ink, Ketcham Manufacturing, Ottawa, ON, Canada) and weighed daily from PND 4 until PND 35. Permanent identification chips (Model IMI-1000, Bio Medic Data Systems, Seaford, DE) were implanted in the pups on PNDs 27–28 under light isoflurane anesthesia.

Testing.
The male and female pups in each litter were assigned to one of four test groups for subsequent behavioral testing. Developmental landmarks (righting reflex, grip strength, negative geotaxis, eye and ear opening, pinna detachment) were measured in all pups. Surface righting and negative geotaxis were evaluated in all offspring on PND 6 and repeated daily until successful completion within 60 sec. Pinna detachment was evaluated starting on PND 6 and repeated daily until the pinna of both ears was completely detached. Eye opening and ear opening were evaluated daily starting on PND 12 and repeated daily until present bilaterally. Grip strength testing was conducted on PNDs 10, 12, and 14 by suspending each pup from a 3-mm wire suspended 30 cm above a 2.5-cm layer of wood shavings. Grip strength was measured as the time (to a maximum of 60 sec) that the pup held itself suspended from the wire.

Reproduction, growth, and development measures included number of litters, number of pups, male/female ratio, uterine implantation sites, litter weights (PNDs 1–4), pup weights (PNDs 4–75), litter mortality (deaths before PND 4), and pup mortality (deaths after PND 4). One male and one female per litter were sacrificed at PNDs 35, 75, and 350 for collection of tissues for systemic toxicology, brain neurochemistry and biomarkers, and thyroid function and residue analysis. One male and one female from one-half of the litters were sacrificed for neuropathology analysis at PND 35, and one male and one female from the remaining litters were sacrificed at PND 70. The dams were sacrificed 7–10 days after weaning for collection of tissues for systemic toxicology, thyroid function, and residue analysis. All animals were sacrificed by guillotine, and trunk blood was collected in SST Vacutainer® tubes (Becton-Dickinson) and allowed to clot at room temperature prior to being placed on ice. Serum was collected by centrifugation at 3000 g for 15 min and frozen at -80°C until analysis. Systemic toxicology, brain biochemistry and biomarkers, neuropathology, and residue analysis results will be reported separately.

Commercially available kits were used to assay serum levels of thyroid stimulating hormone (TSH) (Cat No. RPA5541, Amersham Pharmacia, Piscataway, N.J.), thyroxine, triiodothyronine, and serum triiodothyronine uptake (T4, T3, and T3-UP, respectively; Cat Nos. 06B-254029, -256447, and -237124, respectively; ICN Biomedicals, Aurora, OH). Thyroid follicle histomorphology was determined using ImagePro 4.0 digital image analysis software (Media Cybernetics, Silver Spring, MD) as described in Wade et al. (2002)Go. Briefly, thyroid glands fixed in 10% neutral buffered formalin for at least 10 days were embedded in paraffin, and 5-µm-thick sections were stained by the Periodic-Acid Schiff method. Follicle morphology was determined from two digital pictures of the interior region of the thyroid gland from each animal. Measures included follicle colloid cross-sectional area, roundness [(colloid perimeter)2/4 x (colloid area)]), and aspect ratio (longest diameter/shortest diameter).

Statistical analyses.
Parametric and nonparametric statistical tests were used depending on the specific type of data. Maternal and offspring body weight data and grip strength data were analyzed with a two-way (dose x days) mixed effects analysis of variance (ANOVA) followed by simple effects tests where appropriate. Litter size and implantation sites were analyzed with a one-way ANOVA. Mortality rates and sex ratios were analyzed with nonparametric Kruskal-Wallis ANOVA and followed with pairwise Mann-Whitney U where the overall Kruskal-Wallis ANOVA revealed a significant overall treatment effect. Thyroid data were analyzed with two-way (treatment and cohort) ANOVA. Where ANOVA assumptions could not be satisfied by log or arcsine transformations, nonparametric Wilcoxon/Kruskal-Wallis tests were conducted. Significant ANOVA effects were probed with Tukey/Kramer post hoc tests to identify the specific nature of the treatment effects. Tests of early neurodevelopmental events (eye and ear opening, pinna detachment, negative geotaxis, righting reflex) were recorded as binary data (present/absent) and analyzed using nonparametric Kruskal-Wallis ANOVA. Where treatment effects were detected, follow-up nonparametric tests (Mann-Whitney U or discrete Kruskal-Wallis ANOVA) were conducted to determine where the treatment groups differed. To control for differential mortality rates between dose groups, results from neurobehavioral tests included only data from animals that completed the specific test.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Because body weight increased slightly between the preparation and the delivery of the dosed cookies, the exact doses delivered to the dams were calculated based on the body weight on the day the dose was delivered, the volume delivered to the dosing cookie, and the concentrations of the dosing solutions (see Table 1Go). The total measured mass of the mixture at the highest dose was 13.05 mg/ml. The mean doses averaged over the 45 days of dosing were 0.013, 0.130, 1.30, and 13.08 mg/kg/day. The mean dose delivered to the Aroclor 1254 animals was 14.98 mg/kg/day.


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TABLE 1 Concentrations of Persistent Organochlorine Pollutants in POP Mixture.
 
The highest dose of the mixture (13 mg/kg) decreased maternal weight gain during pregnancy (Fig. 1Go). ANOVA revealed a significant dose by days of interaction (F20,330 = 6.99, p < 0.001), and simple effects tests revealed that the dose affected body weight on GDs 11, 16, and 21 (F5,87 > 3.40, p < 0.008). Contrasts confirmed that maternal weight gain was decreased only in the 13-mg/kg mixture dose group on GDs 11, 16, and 21 (t > 2.70, p < 0.01). The reduced maternal body weight in the dams exposed to the highest dose of the mixture also persisted throughout the lactation period, as revealed by a main effect of dose (F5,83 = 5.81, p < 0.001), but the effect of dose did not vary over lactation days (F20,266, p = 0.217).



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FIG. 1. Mean (± sem) maternal weight gain during gestation and lactation in dams exposed to the chemical mixture or Aroclor 1254.

 
Table 2Go shows the number of litters, litter size, implantation sites, sex ratio, and mortality rates. There were no dose-related effects on litter size (F5,87 =.269, p = 0.929), the number of implantation sites (F5,87 = 2.86, p = 0.767), sex ratio of the offspring ({chi}2,5 = 1.418, p = 0.992), or ratio of pups born to implantation sites (F5,87 = 1.12, p = 0.356). The intermediate mixture dose (1.3 mg/kg) produced a small decrease in gestation period (F5,87 = 2.87, p = 0.019) that was due to an increase in the proportion of litters born by GD 22 (62%) relative to control animals (24%). There was some indication that the highest dose of the mixture may have altered fetal viability. In two of the 14 pregnant females dosed with 13 mg/kg of the mixture, weight losses occurred early in pregnancy followed by an increase in body weight characteristic of pregnancy. In both cases, the number of uterine implantation sites was normal (14 and 17), but only three and four pups were born. One pregnant female dosed with 0.013 mg/kg of the mixture had an unusually small litter (two pups); however, the number of implantation sites was also unusually small (four detectable implantation sites).


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TABLE 2 Reproductive Data and Mortality Rates in Litters Exposed to the Chemical Mixture or Aroclor 1254 from GD 1 to PND 23.
 
As shown in Table 2Go, more than 40% of the pups born to dams dosed with 13 mg/kg of the mixture died by PND 4 with mortalities in 93% of these litters. No other mixture dose affected mortality rates. The 13-mg/kg mixture dose also increased pup mortality after culling litters to eight pups at PND 4 (66% of remaining offspring). Moreover, these mortalities occurred in 93% of the litters, indicating that the high overall mortality rate in this treatment group was not due to mortality in a small subset of the litters in this dose group. No mortalities occurred in any other mixture group after PND 4. Aroclor 1254 produced a slight but nonsignificant increase in mortality rates prior to PND 4 (Mann-Whitney U = 98.5, p = 0.401) and a small increase in mortality rates (14%) after PND 4. Aroclor 1254 produced significantly less mortality than 13 mg/kg of the mixture ({chi}2 = 10.83, p = 0.001), and mortalities in the Aroclor 1254 after PND 4 were distributed over 50% of the litters.

Because of the large differences in mortality between dose groups, the litter weights were corrected to adjust for differential litter mortality by calculating mean litter weight standardized on the number of surviving pups (i.e., mean pup weight/litter). Litter weight increased between PND 1 and PND 4 (F3,83 = 425.4, p < 0.001), and dose significantly altered normalized litter weight gain (F5, 87 = 17.58, p < 0.001). Contrasts revealed that both 13 mg/kg of the mixture and Aroclor 1254 significantly decreased mean normalized litter weights (t > 2.8, p < 0.01) but no other dose affected litter weights.

Both 13 mg/kg of the mixture and Aroclor 1254 reduced offspring weight gain, and the reduction in weight gain increased up to weaning age (Fig. 2Go). ANOVA confirmed the effect of dose (F5,622 = 56.85, p < 0.001) and the dose by age interaction (F30,3077 = 16.33, p < 0.001). Although the females were smaller than the males, there was no indication that gender altered the impact of the dose on body weight (F5,622 = 0.63, p = 0.676), nor was there any significant three-way interaction between dose, gender, and age (F30,3077 = 0.95, p = 0.542). Because of the high mortality in the 13-mg/kg mixture group, and because Aroclor 1254 was used as a positive control treatment, a separate repeated measures ANOVA was conducted on body weight data excluding these groups. There was a marginally significant dose by age interaction (F18,1451 = 1.62, p = 0.049) associated with dose-related differences in weight gain at PNDs 17 and 20 (F3,490 >3.2, p < 0.025), but the mean weight differences between dose groups at these ages were less than 1 g.



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FIG. 2. Mean (± sem) weight gain between PND 5 and weaning at PND 23 in offspring of dams exposed to the chemical mixture or Aroclor 1254 during gestation and lactation. Litter weights in the first four postnatal days were significantly reduced in offspring exposed to 13 mg/kg of the chemical mixture or Aroclor 1254 (data not shown).

 
Figure 3Go shows the weight gain in offspring from PND 25 to 65. Only the animals necropsied at PND 70 or 350 were included in this analysis. Body weight data collected after PND 70 were excluded because the animals were placed on restricted diets (90% free-feeding weights) at PND 70 for subsequent behavioral testing (data reported separately). Mean body weights for five blocks of 10 days were computed (e.g., day 25 weight = mean of PND 21 to PND 30). Weight data for the 13-mg/kg-mixture group was excluded from the analysis even though the mean body weight for this group is included in Figure 3Go for illustrative purposes. Because of the heterogeneity of variance and violations of the circularity assumption, raw body weights were transformed with a log10 transformation and the transformed data analyzed with repeated measures ANOVA. ANOVA confirmed that the males were larger than the females (main effect of gender F1,370 = 919.5, p < 0.001). The reduced body weight in the 13-mg/kg-mixture and Aroclor 1254 groups persisted to at least PND 70. ANOVA confirmed that Aroclor 1254 altered body weight (F4,370 = 39.15, p < 0.001) but also revealed a significant dose by days interaction (F16,1121 = 14.56, p > 0.001). Simple effects tests confirmed that body weight was significantly reduced in Aroclor 1254–treated animals from PND 23 to PND 70 (t > 5, p < 0.001). There was a small (5–8 g) but significant decrease in body weight in animals dosed with 1.3 mg/kg of the mixture at PNDs 35 and 45 (t > 1.9, p < 0.05) but not thereafter (p > 0.10). Though excluded from the statistical analysis, Figure 3Go also shows that 13 mg/kg of the mixture produced a persistent reduction in body weight (40% decrease) relative to the control animals that was larger than the decrease in Aroclor 1254-treated animals (15%). There was no indication that the animals recovered from this weight deficit by PND 70. Finally, there was no indication that the effect of doses differed between males and females (F4,370 = 0.460, p = 0.765).



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FIG. 3. Mean (± sem) weight gain from weaning to PND 70 in female (A) and male (B) offspring exposed to the chemical mixture or Aroclor 1254.

 
Developmental landmarks were affected by both the chemical mixture and Aroclor 1254. There was no indication that pinna detachment was affected since it was present in all dose groups at PND 6. Negative geotaxis was delayed in animals exposed to the 13-mg/kg mixture ({chi}2 = 16.207, p = 0.006). A separate analysis that excluded the 13-mg/kg-mixture group revealed that no other treatment altered the appearance of negative geotaxis ({chi}2 = 2.862, p = 0.581). ANOVA on time to complete negative geotaxis on the day when it occurred revealed no dose (F5,635 = 0.326, p = 0.898), gender (F1,635 = 0.230, p = 0.631) or interaction effects (F5,635 = 1.55, p = 0.181).

Days to occurrence of the righting reflex was also altered by treatments ({chi}2 = 48.936, p < 0.001). Unlike the negative geotaxis, both 13 mg/kg of the mixture and Aroclor 1254 delayed the appearance of the righting reflex. Separate analyses indicated that righting reflex was delayed when either 13 mg/kg of the mixture ({chi}2 = 35.942, p < .001) or Aroclor 1254 ({chi}2 = 28.357, p = < 0.001) was included but not when both were excluded from the analysis.

Exposure to the chemical mixture or Aroclor 1254 delayed ear opening ({chi}2 = 210.3, p < 0.001) (Fig. 4AGo). Except for the lowest mixture dose (0.013 mg/kg), all mixture doses delayed ear opening (p < 0.001), as did Aroclor 1254 (Mann-Whitney U = 4091, p < 0.001). In addition, the highest mixture dose produced a significantly greater delay in ear opening than Aroclor 1254 (Mann-Whitney U = 582, p < 0.001), but Aroclor 1254 produced significantly greater delays than 0.13 or 1.3 mg/kg of the mixture (p < 0.001). The appearance of eye opening was also altered by exposure to the mixture or Aroclor 1254 ({chi}2 = 35.5, p < 0.001) (Fig. 4BGo). A separate Kruskal-Wallis test that excluded both 13-mg/kg-mixture and Aroclor 1254 doses revealed that eye opening was significantly altered by lower doses of the mixture ({chi}2 = 16.32, p = 0.001). Both Aroclor 1254 and 1.3 mg/kg of the mixture accelerated eye opening (p < 0.02), but Aroclor 1254 was more effective (Mann-Whitney U = 4781, p < 0.001). The other mixture doses delayed eye opening, but the effect was marginally significant only in the lowest dose group (Mann-Whitney U = 3.49, p = 0.062).



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FIG. 4. Proportion of offspring exhibiting ear (A) and eye (B) opening by days of age in offspring exposed to the chemical mixture or Aroclor 1254 during gestation and lactation.

 
Grip strength was evaluated at PNDs 10, 12, and 14. Because of the high mortality by PND 4, the 13-mg/kg-mixture dose group was excluded from the analysis. Because grip time data violated the homogeneity of variance assumption (F > 1.9, p < 0.01) and circularity assumptions of ANOVA (Box-M = 687.2, p < 0.001), grip time scores were submitted to a log10 transformation and the transformed data were analyzed with repeated measures ANOVA. As shown in Figure 5Go, grip time increased with age (F2,548 = 429.11, p < 0.001) and was affected by the mixture and Aroclor 1254 (F4,549 = 14.27, p < 0.001). Simple effects tests indicated that 0.013 and 1.3 mg/kg of the mixture and Aroclor 1254 significantly decreased grip time (p < 0.03) (Fig. 5Go). Though excluded from the analysis, 13 mg/kg of the mixture produced the largest decrease in grip time of all of the treatments. There were no effects of gender (F1,549 = 1,75, p = 0.187) nor any dose by gender interactions (F4,549 = 0.57, p = 0.69).



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FIG. 5. Mean (± sem) grip strength (grip time) at PNDs 10, 12, and 14 in offspring exposed to the chemical mixture or Aroclor 1254 during gestation and lactation.

 
Figure 6AGo shows normal thyroid gland histology in a representative control dam characterized by numerous spherical follicles composed of peripheral epithelial layer surrounding a central, acellular lumen filled with thyroglobulin-rich colloid. In animals with toxicant-induced hypothyroxinemia, such as Aroclor 1254-treated dams (Fig. 6CGo), follicles tend to be reduced in volume, less round in cross section, and epithelial cells that show hypertrophy with vacuolated cytoplasm, particularly in the apical region adjacent to the follicle lumen. Dams treated daily with 13 mg/kg of the mixture show changes indicating hypothyroxinemia (Fig. 6BGo). Thyroid glands from dams receiving lower doses of the mixture exhibited morphology similar to the control animals (data not shown). Aroclor 1254 reduced the median follicle cross-sectional area to about one-third of the vehicle treated animals (p > 0.01), decreased the proportion of thyroid follicles with cross-sectional areas greater than 1000 µm2 (p < 0.01) (Fig. 7BGo), and produced a small but significant increase in the index of roundness (p = 0.03) (Fig. 7CGo). The 13-mg/kg dose of the mixture also decreased follicle area, as indicated by both reduced median area (p = 0.018) and reduced proportion of follicles with cross-sectional areas greater than 1000 µm2 (p < 0.01) (Figs. 7AGo and 7BGo, respectively), but did not affect measures of roundness (p = 0.113; Fig. 7CGo). Follicle cross-sectional aspect ratio (longest diameter/shortest diameter) was not significantly altered by any treatment (p = 0.195). Although the data for lower doses suggested that there may be a dose-related reduction in follicle cross-sectional area, this measure did not differ significantly from vehicle-treated animals (p > 0.05).



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FIG. 6. Representative photomicrographs of thyroid morphology in dams exposed to vehicle (A), 13 mg/kg of the mixture (B), and Aroclor 1254 (C). Dams were exposed during gestation and lactation and sacrificed 7–10 days after the litters were weaned. A "C" inside figures indicates acellular lumen filled with colloid, black arrowheads indicate thyroid epithelial cells, and white arrowheads indicate the basement membrane surrounding the follicle. Bar = 100 µm.

 


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FIG. 7. Morphometric measures of thyroid gland structure in dams exposed to the chemical mixture or Aroclor 1254 during gestation and lactation. Measures of cross-sectional area of the colloid include the median colloid area (A) and the proportion of all follicles larger than 1000 m2 (B). Measures of roundness of the colloid cross section include roundness (C) and the aspect ratio (D). Data are reported as mean ±standard deviation. Means with the same letter are not significantly different (p > 0.05).

 
The highest mixture dose (13 mg/kg) and Aroclor 1254 produced comparable decreases in serum T4 levels in dams (p < 0.001) (Table 3AGo), but serum T3, T3 uptake and TSH were not significantly affected by any treatment (p > 0.05). Among PND 35 offspring, 13 mg/kg of the mixture and Aroclor 1254 produced significant reductions in serum T4 and T3 (p < 0.001) but did not affect T3 uptake or TSH (Table 3BGo). Mixture doses between 0.013 to 1.3 mg/kg had no significant effects on serum thyroid measures in offspring (p > 0.05). Males had significantly higher serum levels of T4 than females (p = 0.034), but there was no indication that gender influenced the impact of the mixture or Aroclor on serum thyroid hormone levels (p > 0.05).


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TABLE 3 The Effects of Gestational and Lactational Exposure to the Chemical Mixture on Serum Levels of T4, T3, T3 Uptake, and TSH in Dams (A) 7–10 Days after the Last Dosing, or Offspring Sacrificed at PND 35 (B). Both 13 mg/kg of the Mixture and Aroclor 1254 Reduced Serum T4 and T3 Levels in Both Dams and Offspring
 
As previously mentioned, the 13-mg/kg-mixture dose produced high mortality rates that we had not anticipated based on pilot study results. One other unexpected result observed in the highest mixture dose offspring was apparent facial malformations characterized by a rounded skull and underdeveloped snout and lower jaw. In addition to the rough fur characteristic of unhealthy animals, these offspring also appeared to exhibit softened ligaments. Though eye opening was only slightly delayed in the 13-mg/kg-mixture offspring, eye opening appeared incomplete and most of these pups exhibited red crusty deposits around the eyes. In addition, these anomalies were observed in most surviving offspring in this dose group but none of these anomalies were observed in offspring in any other dose group.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The current study employed Aroclor 1254 as a positive control treatment to ensure that the neurobehavioral measurements employed were sensitive to disruption and to compare the impact of a known neurotoxic PCB mixture, Aroclor 1254, against a mixture that mimics the profile of chemicals found in human blood. As expected, Aroclor 1254 affected a number of reproductive, developmental, and neurobehavioral measures. Aroclor 1254 reduced offspring body weight, and this decrease appears to persist until adulthood. This reduced offspring body weight occurred in the absence of significant effects on either maternal body weight or litter size. Aroclor 1254, however, did produce a slight increase in offspring mortality after PND 4. These results are consistent with previous reports on the effect of Aroclor 1254 where a comparable dose range and dosing regimen was employed. For instance, others have reported that exposure to 6–20 mg/kg/day of Aroclor 1254 between GD 6 to PND 21 produces decreases in offspring body weight and increases in offspring mortality but no alterations in maternal body weight or litter sizes (Bushnell et al., 2002Go; Geller et al., 2001Go; Goldey and Crofton, 1998Go; Goldey et al., 1995aGo; Hany et al., 1999bGo).

Aroclor 1254 also altered the appearance of a number of neurodevelopmental landmarks, including delayed righting reflex and ear opening, but accelerated eye opening. The acceleration of eye opening has been reported previously by others and appears to be dose-related. For example, 8 mg/kg/day of Aroclor 1254 from GD 9 to PND 21 accelerates eye opening (Goldey and Crofton, 1998Go; Goldey et al., 1995aGo); however, 6 mg/kg/day over a comparable dosing period does not appear to alter eye opening (Overmann et al., 1987Go). The occurrence of accelerated eye opening following gestational and lactational exposure to Aroclor 1254 appears to occur within a small dose range and under limited conditions (e.g., doses between 8 and 15 mg/kg/day, absence of maternal weight loss, offspring weight loss that persists to adulthood, and limited offspring mortality). At lower doses of Aroclor 1254, where eye opening was not affected, maternal body weight was also unaffected and decreases in offspring weight gain appeared to be transient. Aroclor 1254 also reduced grip strength, and there was no indication that this effect abated over the testing period. Indeed, there was some indication that the effect of Aroclor 1254 on grip strength actually increased over the 3 days of testing.

Exposure to the chemical mixture also altered reproductive, developmental, and neurobehavioral endpoints. The highest mixture dose (13 mg/kg) was toxic to both the dams and offspring, but lower doses of the mixture produced few effects in the dams or on offspring mortality and weight gain. The highest dose of the mixture decreased maternal body weight, offspring weight gain, and survival. Two dams in the 13-mg/kg-mixture group delivered small litters despite a normal number of uterine implantation sites. The combination of a normal number of implantation sites, unusual gestational weight gain pattern, and small litter size in these two litters suggests that the highest mixture dose may have produced fetal resorptions. Whether this was an indirect result of decreased body weight or a direct impact of the highest mixture dose is not clear.

Exposure to the chemical mixture altered a number of early neurodevelopmental measures. The 13-mg/kg-mixture dose altered negative geotaxis, the righting reflex, ear opening, and grip strength. It is difficult to attribute these changes to specific neurotoxicological effects since they may be secondary to the nonspecific toxicity, as indicated by the decreased maternal and offspring weight gains and high offspring mortality. Some early developmental impairments were evident at lower doses of the mixture where neither maternal toxicity nor offspring weight gain or mortality were affected. For instance, 1.3 mg/kg of the mixture produced modest alterations in ear opening, eye opening, and grip strength. The acceleration in eye opening produced by 1.3 mg/kg of the mixture was comparable to the acceleration produced by Aroclor 1254. These alterations at lower doses of the chemical mixture provide evidence that the nonlethal doses of the mixture produces mild effects on early neurodevelopmental landmarks even though these measures are not known to be particularly sensitive neurotoxicological endpoints.

Aroclor 1254 is known to alter thyroid function (Porterfield, 2000Go). Similarly, a number of the chemical components of the mixture are also known to alter thyroid function when administered individually (e.g., hexachlorobenzene, chlordanes, PCBs). As such, we had anticipated that both Aroclor 1254 and the mixture would alter thyroid hormone function in the dams as well as their offspring. Results for both serum hormone levels and gland histomorphology indicate that both Aroclor 1254 and 13 mg/kg of the mixture caused frank hypothyroxinemia in the pregnant dams. The serum measures of thyroid hormone status reported in the current study are widely recognized as useful indicators of thyroid function in humans and animals (DeVito et al., 1999Go). Further, while thyroid gland histopathology is recognized as a reliable indicator of toxicity to the hypothalamic–pituitary–thyroid axis (Capen, 1992Go), serum thyroid hormone levels and thyroid histopathology can be considered only as surrogate indicators of thyroid hormone status. Thyroid hormone action within the brain is dependent on a variety of factors, including T4 crossing the blood brain barrier, de-iodination of T4 to T3 in target brain nuclei, ratios of thyroid hormone receptor co-activators to co-inhibitors, etc. (Brouwer et al., 1998Go; Porterfield and Hendrich, 1993Go). Thyroid effects directly relevant to neurodevelopmental toxicology can only be determined by examining levels of expression of thyroid hormone-dependent genes in target tissues during critical developmental windows. Because these were not quantified in this study, it remains possible that the mixture may have caused regional hypothyroidism within specific brain nuclei involved in central nervous system (CNS) development and in the regulation of behavior. Moreover, thyroid hormone levels were evaluated in offspring at PND 35, and this does not necessarily reflect thyroid status during the developmental period critical for neurological development (between GD 10 and PND 20) (Oppenheimer and Schwartz, 1997Go; Porterfield and Hendrich, 1993Go). It is possible that the mixture induced hypothyroidism during this period but was not detected.

Despite these limitations, it appears unlikely that altered thyroid function plays a major role in the developmental effects of the chemical mixture for a number of reasons. First, though Aroclor 1254 and 13 mg/kg of the mixture produced similar effects on thyroid hormone levels and thyroid histopathology in the dams, the mixture was considerably more lethal than Aroclor 1254. Second, some developmental disturbances observed in the 13-mg/kg-mixture group are inconsistent with hypothyroidism. Specifically, hypothyroidism is known to delay eye opening (Brosvic et al., 2002Go; Goldey et al., 1995bGo), and gestational and lactational thyroxine treatment accelerates eye opening (Brosvic et al., 2002Go; Goldey and Crofton, 1998Go). Despite similar effects on thyroid hormone levels and histopathology in dams, Aroclor 1254 accelerated eye opening but 13 mg/kg of the mixture delayed eye opening. Additionally, the mixture was considerably more potent in delaying ear opening than Aroclor 1254. Taken together, the similar effects of Aroclor 1254 and the 13-mg/kg dose of the mixture on thyroid function and morphology combined with contrasting effects on mortality, growth, and early developmental measures indicate that these effects are unlikely to be mediated exclusively by thyroid mechanisms.

Commercial Aroclor mixtures are frequently used as a surrogate for human PCB exposure patterns in the evaluation of the toxicity of PCBs (Hany et al., 1999bGo; Herr et al., 1996Go; Kodavanti et al., 1998Go). The results of the current study suggest that the use of commercial mixtures of PCB like Aroclor 1254 may model human PCB exposures but may not provide adequate estimates of the toxic effects of the broader range of contaminants found in humans. While we found similarities between Aroclor 1254 and the highest mixture dose (i.e., reduced offspring body weight, delayed righting reflex, delayed ear opening, reduced grip strength, and maternal hypothyroxinemia), exposure to the mixture produces a different pattern of effects than Aroclor 1254. For instance, while 13 mg/kg of the mixture produced decreases in maternal gestational and lactational weight gain, high mortality rates, and some evidence of fetal resorptions, Aroclor 1254 has no impact on maternal weight gain, produced small increases in mortality rates, less pronounced offspring weight loss, and no indications of fetal resorptions. Moreover, facial malformations were evident in most offspring exposed to 13 mg/kg of the mixture but not in any Aroclor 1254-exposed offspring. Taken together, our results indicate that the toxicity of developmental exposure to Aroclor 1254 differs considerably from the toxicity of the complex mixture that more completely mimics human exposure profiles. The mixture used in the current study, however, contains both PCBs and organochlorine pesticides and is more similar to the chemical profile associated with human body burden. The total dose of all the PCB congeners in the 13-mg/kg-mixture dose was about 5 mg/kg/day, compared with 15 mg/kg in the Aroclor 1254-dose group. Because the mixture contains about 33% of the PCB dose of Aroclor 1254, it seems unlikely that the greater toxicological effects of 13 mg/kg of the mixture is related to the total dose of PCB congeners in the mixture. It is possible that differences between the PCB congener profiles in the Aroclor 1254 and the chemical mixture may account for the difference in toxicity, or they may be related to the inclusion of organochlorine pesticides in the mixture. Regardless of the specific differences between the mixture and Aroclor 1254, it appears that the toxicity of perinatal Aroclor 1254 does not adequately mimic the toxicity after perinatal exposure to a chemical mixture that simulates human blood levels.

There are a number of difficulties associated with conducting studies of complex mixtures in animals that should be noted. Because of toxicokinetic factors (absorption, metabolism, distribution, and elimination), the contaminant profile for in utero exposure is likely to be somewhat different than the mixture profile and thus human blood profile that served as the basis for the mixture used in the current study. In addition, the relationship between the blood levels produced in animals in this study and human blood levels cannot be precisely determined until planned blood residue analyses are completed. These results will also be important in determining the relationship between the doses used in this study and the contaminant levels in human blood. The high toxicity (maternal and offspring weight loss and offspring mortality) found with the highest mixture dose used in this study suggests that this dose produces considerably higher blood levels than has been observed in human populations. However, human intake of most contaminants contained in the mixture have been estimated previously (Health Canada, 1998Go). Using these exposure estimates, and based on an estimated 50% absorption by the mother and 50% of the absorbed contaminants distributed to the fetuses, the lowest dose used in the current study is about 1.7 times the estimated intake in human infants between 0 and 7 months of age. Thus, the dose range that we selected does produce estimated intakes comparable to the estimated human intake, but the high early developmental toxicity produced by the highest mixture dose produces estimated intakes about 1000 times more than current human intake. These estimates should be treated cautiously, however, until blood levels measures are available.

Though not our primary objective, it should be noted that we found considerable differences in the toxicity among offspring between dosing from GD 11 to GD 17 and dosing from GD 1 to PND 23. That a longer dosing period produces greater toxicity is not especially surprising, but the complete absence of any indications of increased mortality or altered body weight in either the dams or offspring after Aroclor 1254 and 13 mg/kg of the mixture from GDs 11–17 contrasts sharply with the 80% overall mortality rates, maternal weight loss, and persistent offspring weight loss in offspring after exposure to the same doses from GD 1 to PND 23. Differences in the effects on offspring mortality and weight gain also occurred after exposure to Aroclor 1254, though these were less dramatic than for the chemical mixture. This differential toxicity associated with the two dosing regimens illustrates the critical importance of the dosing regimen in assessing developmental effects of chemical exposures. As such, studies that examine the neurotoxicological effects of chemical exposure with a restricted perinatal dosing period during pregnancy in order to identify critical exposure periods may not provide realistic estimates of developmental neurotoxicity in humans where exposure occurs over the entire gestational and lactational periods. This is of particular importance in using animal toxicity data from developmental studies to estimate human health risks from perinatal exposure to mixtures of chemicals.

In summary, developmental exposure to a mixture of PCBs and organochlorine pesticides based on human blood levels produces greater toxicity than a comparable dose of the commercial PCB mixture Aroclor 1254. In addition to affecting maternal weight gains, the mixture produced significant and persistent reductions in offspring weight, high mortality rates, altered early neurobehavioral function, and produced facial malformations in offspring. Since comparable alterations in thyroid status were evident in Aroclor 1254- and mixture-exposed animals, altered thyroid function is not likely responsible. In addition, differences in developmental dosing periods substantially affected the toxicity of the chemical mixture and, to a lesser degree, Aroclor 1254, indicating that limited developmental exposure may not provide realistic assessments of developmental toxicity of environmental chemicals. Finally, our results of in utero and lactational exposure to the chemical mixture suggest that current human exposure is below toxic levels for early developmental measures. Further work is currently in progress to characterize the longer term effects of this chemical mixture on more complex neurotoxicological and systemic endpoints.


    ACKNOWLEDGMENTS
 
This study was supported by a grant from the Toxic Substances Research Initiative from Health Canada and Environment Canada (TSRI # 209). We thank Bruce Martin, Susan Kelly, Kelly Brennan and Yasmine Dirieh for the excellent technical assistance.


    NOTES
 
This paper has been reviewed by Health Canada and approved for publication. Approval for publication does not signify that the contents reflect the views or policy of Health Canada.

1 To whom correspondence should be addressed at Health Canada, Environmental Health Sciences Bureau, Room B35, Environmental Health Centre, P.L. 0803B,Ottawa, Ontario K1A 0L2, Canada. Fax: (613) 957-8800. E-mail: wayne_j_bowers{at}hc-sc.gc.ca Back


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