* Systemic Toxicology and Pharmacokinetics Section, Healthy Environments and Consumer Safety Branch, and
Toxicology Research Division, Health Products and Foods Branch, Health Canada, Ottawa, Ontario K1A 0L2, Canada
Received July 9, 2003; accepted September 12, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: behavior; PCB; neurotoxicity; chemical mixture; organochlorine; thyroid; DDT; developmental toxicity; Aroclor 1254; Great Lakes; thyroid.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1998; Yang, 1998
). 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, 1999
) and PCBs and pesticides (Carr et al., 2002
). 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., 2001
). 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, 2000; Porterfield and Hendrich, 1993
). 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., 2000
; Brouwer et al., 1998
; van Raaij et al., 1991
), we measured biomarkers of thyroid function in dosed dams and offspring.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 Canadas Institutional Animal Care Committee prior to the start of the study. Ninety-three nulliparious female Sprague Dawley rats (200230 g) and 38 male Sprague Dawley rats (315350 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 males 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 males 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 2728 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 14), pup weights (PNDs 475), 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 710 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). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
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. 2). 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.
|
|
Days to occurrence of the righting reflex was also altered by treatments (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 (
2 = 35.942, p < .001) or Aroclor 1254 (
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 (2 = 210.3, p < 0.001) (Fig. 4A
). 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 (
2 = 35.5, p < 0.001) (Fig. 4B
). 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 (
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).
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, 1998; Goldey et al., 1995a
); however, 6 mg/kg/day over a comparable dosing period does not appear to alter eye opening (Overmann et al., 1987
). 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, 2000). 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., 1999
). Further, while thyroid gland histopathology is recognized as a reliable indicator of toxicity to the hypothalamicpituitarythyroid axis (Capen, 1992
), 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., 1998
; Porterfield and Hendrich, 1993
). 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, 1997
; Porterfield and Hendrich, 1993
). 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., 2002; Goldey et al., 1995b
), and gestational and lactational thyroxine treatment accelerates eye opening (Brosvic et al., 2002
; Goldey and Crofton, 1998
). 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., 1999b; Herr et al., 1996
; Kodavanti et al., 1998
). 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, 1998). 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 1117 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 |
---|
![]() |
NOTES |
---|
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
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bernhoft, A., Nafstad, I., Engen, P., and Skaare, J. U. (1994). Effects of pre- and postnatal exposure to 3,3'4,4',5-pentachlorobiphenyl on physical development, neurobehavior and xenobiotic metabolizing enzymes in rats. Environ. Toxicol. Chem. 13, 15891597.[ISI]
Bondy, G. S., Newsome, W. H., Armstrong, C. L., Suzuki, C. A., Doucet, J., Fernie, S., Hierlihy, S. L., Feeley, M. M., and Barker, M. G. (2000). Trans-Nonachlor and cis-nonachlor toxicity in Sprague-Dawley rats: Comparison with technical chlordane. Toxicol. Sci. 58, 386398.
Brosvic, G. M., Taylor, J. N., and Dihoff, R. E. (2002). Influences of early thyroid hormone manipulations. Delays in pup motor and exploratory behavior are evident in adult operant performance. Physiol. Behav. 75, 697715.[CrossRef][ISI][Medline]
Brouwer, A., Morse, D. C., Lans, M. C., Schuur, A. G., Murk, A. J., Klasson-Wehler, E., Bergman, A., and Visser, T. J. (1998). Interactions of persistent environmental organohalogens with the thyroid hormone system: Mechanisms and possible consequences for animal and human health. Toxicol. Ind. Health 14, 5984.[ISI][Medline]
Bushnell, P. J., Moser, V. C., MacPhail, R. C., Oshiro, W. M., Derr-Yellin, E. C., Phillips, P. M., and Kodavanti, P. R. (2002). Neurobehavioral assessments of rats perinatally exposed to a commercial mixture of polychlorinated biphenyls. Toxicol. Sci. 68, 109120.
Capen, C. C. (1992). Pathophysiology of chemical injury of the thyroid gland. Toxicol. Lett. 6465, 381388.[CrossRef]
Carpenter, D. O., Arcaro, K. F., Bush, B., Niemi, W. D., Pang, S., and Vakharia, D. D. (1998). Human health and chemical mixtures: An overview. Environ. Health Perspect. 106, 12631270.[ISI][Medline]
Carr, R. L., Richardson, J. R., Guarisco, J. A., Kachroo, A., Chambers, J. E., Couch, T. A., Durunna, G. C., and Meek, E. C. (2002). Effects of PCB exposure on the toxic impact of organophosphorus insecticides. Toxicol. Sci. 67, 311321.
Cole, D. C., Sheeshka, J., Murkin, E. J., Kearney, J., Scott, F., Ferron, L. A., and Weber, J. P. (2002). Dietary intakes and plasma organochlorine contaminant levels among Great Lakes fish eaters. Arch. Environ. Health 57, 496509.[ISI][Medline]
Crofton, K. M., and Rice, D. C. (1999). Low-frequency hearing loss following perinatal exposure to 3,3',4,4',5- pentachlorobiphenyl (PCB 126) in rats. Neurotoxicol. Teratol. 21, 299301.[CrossRef][ISI][Medline]
Daly, H. B., Hertzler, D. R., and Sargent, D. M. (1989). Ingestion of environmentally contaminated Lake Ontario salmon by laboratory rats increases avoidance of unpredictable aversive nonreward and mild electric shock. Behav. Neurosci. 103, 13561365.[CrossRef][ISI][Medline]
DeVito, M., Biegel, L., Brouwer, A., Brown, S., Brucker-Davis, F., Cheek, A. O., Christensen, R., Colborn, T., Cooke, P., Crissman, J., et al. (1999). Screening methods for thyroid hormone disruptors. Environ. Health Perspect. 107, 407415.[ISI][Medline]
Geller, A. M., Oshiro, W. M., Haykal-Coates, N., Kodavanti, P. R., and Bushnell, P. J. (2001). Gender-dependent behavioral and sensory effects of a commercial mixture of polychlorinated biphenyls (Aroclor 1254) in rats. Toxicol. Sci. 59, 268277.
Goldey, E. S., and Crofton, K. M. (1998). Thyroxine replacement attenuates hypothyroxinemia, hearing loss, and motor deficits following developmental exposure to Aroclor 1254 in rats. Toxicol. Sci. 45, 94105.[Abstract]
Goldey, E. S., Kehn, L. S., Lau, C., Rehnberg, G. L., and Crofton, K. M. (1995a). Developmental exposure to polychlorinated biphenyls (Aroclor 1254) reduces circulating thyroid hormone concentrations and causes hearing deficits in rats. Toxicol. Appl. Pharmacol. 135, 7788.[CrossRef][ISI][Medline]
Goldey, E. S., Kehn, L. S., Rehnberg, G. L., and Crofton, K. M. (1995b). Effects of developmental hypothyroidism on auditory and motor function in the rat. Toxicol. Appl. Pharmacol. 135, 6776.[CrossRef][ISI][Medline]
Hany, J., Lilienthal, H., Roth-Harer, A., Ostendorp, G., Heinzow, B., and Winneke, G. (1999a). Behavioral effects following single and combined maternal exposure to PCB 77 (3,4,3',4'-tetrachlorobiphenyl) and PCB 47 (2,4,2',4'- tetrachlorobiphenyl) in rats. Neurotoxicol. Teratol. 21, 147156.[CrossRef][ISI][Medline]
Hany, J., Lilienthal, H., Sarasin, A., Roth-Harer, A., Fastabend, A., Dunemann, L., Lichtensteiger, W., and Winneke, G. (1999b). Developmental exposure of rats to a reconstituted PCB mixture or Aroclor 1254: Effects on organ weights, aromatase activity, sex hormone levels, and sweet preference behavior. Toxicol. Appl. Pharmacol. 158, 231243.[CrossRef][ISI][Medline]
Health Canada (1998) Persistent environmental contaminants and the Great Lakes Basin population: An exposure assessment, Minister of Public Works and Government Services Canada, Cat No H462/98218E.
Herr, D. W., Goldey, E. S., and Crofton, K. M. (1996). Developmental exposure to Aroclor 1254 produces low-frequency alterations in adult rat brainstem auditory evoked responses. Fundam. Appl. Toxicol. 33, 120128.[CrossRef][ISI][Medline]
Jacobson, J. L., and Jacobson, S. W. (1997). Evidence for PCBs as neurodevelopmental toxicants in humans. Neurotoxicology 18, 415424.[ISI][Medline]
Jacobson, J. L., Jacobson, S. W., and Humphrey, H. E. (1990). Effects of exposure to PCBs and related compounds on growth and activity in children. Neurotoxicol. Teratol. 12, 319326.[CrossRef][ISI][Medline]
Kearney, J. P., Cole, D. C., Ferron, L. A., and Weber, J. P. (1999). Blood PCB, p,p'-DDE, and mirex levels in Great Lakes fish and waterfowl consumers in two Ontario communities. Environ. Res. 80, S138S149.[CrossRef][ISI][Medline]
Kodavanti, P. R., Derr-Yellin, E. C., Mundy, W. R., Shafer, T. J., Herr, D. W., Barone, S., Choksi, N. Y., MacPhail, R. C., and Tilson, H. A. (1998). Repeated exposure of adult rats to Aroclor 1254 causes brain region-specific changes in intracellular Ca2+ buffering and protein kinase C activity in the absence of changes in tyrosine hydroxylase. Toxicol. Appl. Pharmacol. 153, 186198.[CrossRef][ISI][Medline]
Kodavanti, P. R., Kannan, N., Yamashita, N., Derr-Yellin, E. C., Ward, T. R., Burgin, D. E., Tilson, H. A., and Birnbaum, L. S. (2001). Differential effects of two lots of aroclor 1254: Congener-specific analysis and neurochemical end points. Environ. Health Perspect. 109, 11531161.[ISI][Medline]
Koopman-Esseboom, C., Morse, D. C., Weisglas-Kuperus, N., Lutkeschipholt, I. J., van der Paauw, G. C., Tuinstra, L. G., Brouwer, A., and Sauer, P. J. (1994). Effects of dioxins and polychlorinated biphenyls on thyroid hormone status of pregnant women and their infants. Pediatr. Res. 36, 468473.[Abstract]
Koopman-Esseboom, C., Weisglas-Kuperus, N., de Ridder, M. A., van der Paauw, G. C., Tuinstra, L. G., and Sauer, P. J. (1996). Effects of polychlorinated biphenyl/dioxin exposure and feeding type on infants mental and psychomotor development. Pediatrics 97, 700706.[Abstract]
Kosatsky, T., Przybysz, R., Shatenstein, B., Weber, J. P., and Armstrong, B. (1999a). Contaminant exposure in Montrealers of Asian origin fishing the St. Lawrence River: exploratory assessment. Environ. Res. 80, S159S165.[CrossRef][ISI][Medline]
Kosatsky, T., Przybysz, R., Shatenstein, B., Weber, J. P., and Armstrong, B. (1999b). Fish consumption and contaminant exposure among Montreal-area sportfishers: Pilot study. Environ. Res. 80, S150S158.[CrossRef][ISI][Medline]
Lilienthal, H., and Winneke, G. (1991). Sensitive periods for behavioral toxicity of polychlorinated biphenyls: determination by cross-fostering in rats. Fundam. Appl. Toxicol. 17, 368375.[ISI][Medline]
Oppenheimer, J. H., and Schwartz, H. L. (1997). Molecular basis of thyroid hormone-dependent brain development. Endocr. Rev. 18, 462475.
Overmann, S. R., Kostas, J., Wilson, L. R., Shain, W., and Bush, B. (1987). Neurobehavioral and somatic effects of perinatal PCB exposure in rats. Environ. Res. 44, 5670.[ISI][Medline]
Patandin, S., Koopman-Esseboom, C., de Ridder, M. A., Weisglas-Kuperus, N., and Sauer, P. J. (1998). Effects of environmental exposure to polychlorinated biphenyls and dioxins on birth size and growth in Dutch children. Pediat.r Res. 44, 538545.[Abstract]
Porterfield, S. P. (2000). Thyroidal dysfunction and environmental chemicalsPotential impact on brain development. Environ. Health Perspect. 108(Suppl. 3), 433438.[ISI][Medline]
Porterfield, S. P., and Hendrich, C. E. (1993). The role of thyroid hormones in prenatal and neonatal neurological developmentCurrent perspectives. Endocr. Rev. 14, 94106.[ISI][Medline]
Schantz, S. L., Seo, B. W., Wong, P. W., and Pessah, I. N. (1997). Long-term effects of developmental exposure to 2,2',3,5',6- pentachlorobiphenyl (PCB 95) on locomotor activity, spatial learning and memory and brain ryanodine binding. Neurotoxicology 18, 457467.[ISI][Medline]
van Raaij, J. A., van den Berg, K. J., Engel, R., Bragt, P. C., and Notten, W. R. (1991). Effects of hexachlorobenzene and its metabolites pentachlorophenol and tetrachlorohydroquinone on serum thyroid hormone levels in rats. Toxicology 67, 107116.[CrossRef][ISI][Medline]
Wade, M. G., Parent, S., Finnson, K. W., Foster, W., Younglai, E., McMahon, A., Cyr, D. G., and Hughes, C. (2002). Thyroid toxicity due to subchronic exposure to a complex mixture of 16 organochlorines, lead, and cadmium. Toxicol. Sci. 67, 207218.
Yang, R. S. (1998). Some critical issues and concerns related to research advances on toxicology of chemical mixtures. Environ. Health Perspect. 106 (suppl. 4), 10591063.[ISI][Medline]