Abstract
A developmental neurotoxicity study was conducted to generate additional data on the potential functional and morphological hazard to the central nervous system caused by ammonium perchlorate in offspring from in utero and lactation exposure. Female Sprague-Dawley rats (23 to 25/group) were given continuous access to 0 (carrier), 0.1, 1.0, 3.0, and 10.0 mg/kg-day perchlorate in the drinking water beginning 2 weeks prior to mating and continuing through day 10 of lactation for the behavioral function assessment or given continuous access to 0 (carrier), 0.1, 1.0, 3.0, and 30.0 mg/kg-day beginning on gestation day 0 and continuing through day 10 of lactation for neurodevelopment assessments. Motor activity was conducted on postpartum days 14, 18, and 22 and juvenile brain weights, neurohistopathological examinations, and regional brain morphometry were conducted on postpartum days 10 and 22. This research revealed a sexually dimorphic response, with some brain regions being larger in perchlorate-treated male rats than in comparable controls. Even so, there was no evidence of any obvious exposure-related effects on male rat brain weights or neuropathology. The most consistent exposure-related effect in the male pups was on the thickness of the corpus callosum, with both the right- and left-sided measures of the thickness of this white matter tract being significantly greater for the male pups in the 0.1 and 1.0 mg/kg-day exposure groups. The behavioral testing suggests prenatal exposure to ammonium perchlorate does not affect the development of gross motor movements in the pups.
Neurobehavioral tests are regularly used to screen for anomalies in brain development and function. Performance is quantified, and these scores can be used to infer the integrity of neural systems related to the specific task. These tests are particularly useful as animal models of human behavior and exposure in order to evaluate effects of potentially harmful substances.
In a previous developmental neurotoxicology screen (York 1998; York et al. 2004), offspring were exposed to ammonium perchlorate (AP) in utero and during the neonatal period at 0.1, 1.0, 3.0, and 10.0 mg/kg-day via in the maternal drinking water. In that study, AP had no effect on maternal body weight, feed consumption, clinical or necropsy observations, or sexual maturation of pups at exposures as high as 10.0 mg/kg-day. There were also no overall learning and memory effects in the offspring as evaluated by passive avoidance and the swimming water-maze, or sensory and motor skills as evaluated by motor activity or auditory startle. Motor activity was evaluated on postpartum days (PPDs) 14, 18, 22, and 59 using an automated apparatus (Foss and Lochry 1991) consisting of a passive infrared sensor mounted on the outside of a stainless-steel wire-bottom cage. Motor activity, expressed as overall number of movements and time spent in movement, was not significantly increased; however, examination of the time spent in movement on PPD 14 was significantly increased for the male pups in block 5 at the 3.0 mg/kg-day maternal exposure group and significantly decreased for the female pups in block 1 for the 1.0 mg/kg-day maternal exposure. The time spent in movement on PPD 18 was also significantly increased for the male pups in block 1 for the 0.1 mg/kg-day maternal exposure group.
Juvenile and adult brain weights, brain morphometry, and neuropathology were also assessed by York et al. (1998, 2004), with no overall adverse findings. Examination of the size of the brain region of the PPD 12 pups did reveal a significant increase (23.3%) in the coronal-sectional thickness of the corpus callosum in the female pups in the 10.0 mg/kg-day maternal exposure group as the only statistical finding. The thickness of the corpus callosum in the male pups in the 10.0 mg/kg-day maternal exposure group was also increased (30.8%), although this was not statistically significant.
Based on these motor activity and brain morphometry findings, additional studies were recommended by the Environmental Protection Agency (EPA) (US EPA 2002) to better refine these effects using a different method of evaluating motor activity at the same exposure levels, and another developmental neurotoxicology study using a higher and a lower exposure level. This work was to provide scientific support and a rationale for hazard identification, as well as extend the dose-response data to determine a reference dose (RfD) based on the effects observed in both studies.
In the present research, spontaneous locomotor activity of rat pups was used to evaluate potential neurotoxic insults to the developing rat brain to perchlorate. This research was conducted at the Air Force Research Laboratory, Wright Patterson Air Force Base, Ohio, using a different method of measuring motor activity (infrared photobeam breaks) from that had been used in the original work (infrared motion detector; York 1998; York et al. 2004). The testing days (lactation days (DLs) 14, 18, and 22) were the same developmental ages as the previous work and the exposure levels were the same (0.1, 1.0, 3.0, and 10.0 mg/kg-day). Female rats were treated for 2 weeks prior to gestation through day 10 of lactation (DL 10) with one of 5 doses of AP in drinking water.
The neuropathology component of this study was conducted to provide additional brain morphometric data that may correlate with thyroid data (companion paper) and histological changes present within the same experimental population. This research was conducted in Pennsylvania at the Charles River Laboratories’ Preclinical facility; at the site where the original work was performed (York 1998; York et al. 2004). The brain collection days, gestation day (DG) 21 and PPDs 5, 10, and 22, were the same developmental ages as the previous work and the exposure levels were two of the same exposures plus one additional lower and one additional higher exposure levels (0.01, 0.1, 1.0, and 30.0 mg/kg-day).
MATERIALS AND METHODS
Test Material
Ammonium Perchlorate (CAS no. 7790-98-9), 99.8% purity, was obtained from Aldrich Chemical Company, Inc. (Milwaukee, Wisconsin, lot 03907LF) and used at both sites. Test formulations of AP in reverse osmosis membrane processed deionized water (R.O. deionized water) were prepared at least weekly, stored refrigerated, and dosage solutions brought to room temperature prior to usage. Test drinking solutions yielded the target doses of 0 (carrier), 0.01, 0.1, 1.0, and 30.0 mg/kg-day based on actual weekly water consumption for the neurodevelopment portion of the research (Pennsylvania site). Target doses of 0 (carrier), 0.1, 1.0, 3.0, and 10.0 mg/kg-day were used in the behavioral portion of the research (Ohio site). During the exposure period, rats were given continual access to either R.O. deionized water (carrier control group) or AP in R.O. deionized water (test exposure groups) as drinking water. Dosing concentrations were monitored and confirmed on a regular basis using ion chromatography conducted at the Air Force Research Laboratory (AFRL) for both research sites.
Animals and Husbandry
For the neurobehavioral portion of the research performed in Pennsylvania, female Sprague-Dawley [Crl:CD(SD)IGS BR VAF/Plus] rats were supplied by Charles River Laboratories, Inc. (Raleigh, North Carolina). Male rats of the same source and strain were used only as breeders. The female rats were 72 days of age at arrival and weighed 187 to 249 g. After a 2-week acclimation period, 345 mated female rats (evidence of spermatozoa observed in a vaginal smear or a copulatory plug observed in situ) were randomly assigned to one of five exposure groups, 23 to 25 rats per exposure group, and considered to be GD 0. Rats were provided ad libitum Certified Rodent Chow 5002 (PMI Nutritional International, St. Louis, Missouri) from individual feeders during the exposure period. The Sprague-Dawley rat was selected because this strain (1) has been demonstrated to be sensitive to developmental toxicants; (2) is widely used throughout the industry for nonclinical studies of developmental toxicity; and (3) was used in previous developmental neurotoxicology studies. All cage sizes and housing conditions for both sites were in compliance with the
For the behavioral portion of the research conducted in Ohio, 110 adult virgin female Sprague Dawley (Crl:CD BF VAF/Plus) rats and 37 adult male Sprague-Dawley rats (for breeding purposes) were purchased from Charles River Laboratories, Wilmington, MA. Following quarantine, dams were randomly assigned to one of the five dosing groups and dosed for 2 weeks prior to mating with breeder males. Exposure continued through to DL 10. Breeder males were randomly paired with a female, and both were placed in a standard home cage with breeding grids placed on the bottom in replacement of bedding. Each morning of the breeding period, the cages were surveyed for vaginal plugs. If one was observed, the date was recorded as day DG 1, the male was returned to his home cage and the female placed into a new cage with clean bedding. Each dam and delivered litter was housed in a common nesting box during the postpartum period. Rats were provided ad libitum Harlan Teklad Certified Rodent Diet from individual feeders and ad libitum water (purified to ≥18.0 megaohm-cm resistivity) during the exposure period. Iodine in the feed was 2.46 mg/kg.
Experimental Design
For the neurobehavioral portion of the research conducted in Pennsylvania, female rats were given continual access to ammonium perchlorate in drinking water, or nonchlorinated R.O. water, beginning on DG 0 and continuing through DL 10. The US EPA guidelines (1996) recommend starting exposure on DG 6; in this study, the rats were exposed earlier in order to ensure that the dams had achieved a hypothyroid condition. Target exposures were 0 (carrier), 0.01, 0.1, 1.0, or 30.0 mg/kg-day.
The study conducted in Pennsylvania was divided into four subsets in order to facilitate study management and tissue collections. The brain and behavior effects are reported in this manuscript and the respective thyroid and reproductive effects separately in the preceding companion article. In the first subset, dams were sacrificed on DG 21 and maternal blood, thyroid, and brain samples collected. In the second subset, dams were sacrificed on DL 10, and maternal blood, thyroid, and brain samples were collected. Blood (pooled by litter), thyroid, and brain samples were collected from culled pups on PPD 5 and from the remaining PPD 10 pups. In the third subset, dams were sacrificed on DL 22 and maternal blood and thyroid samples were collected. Blood (pooled by sex and litter), thyroid, and brain samples were collected from culled pups on PPD 5 and from the remaining PPD 22 pups. The fourth subset of the study was a standard rat developmental toxicity study and this has been previously reported (York et al. 2003).
The study conducted in Ohio, randomly assigned the dams to one of five dosing groups. Ammonium perchlorate was dissolved in their drinking water at specific concentrations so that dams received doses of 0, 0.1, 1.0, 3.0, or 10.0 mg/kg-day. Dosing concentrations were monitored and confirmed on a regular basis using ion chromatography. Dam body weights and amount of water consumed were monitored on a daily basis (excluding breeding days) to ensure close approximations of the target doses. Dams were dosed for 2 weeks prior to mating with the breeder males, and through to PPD 10. For breeding purposes, individual males were randomly paired with each female and both were placed in a standard home cage with breeding grids placed on the bottom in the place of bedding. Every morning the cages were surveyed for vaginal plugs. If one was observed, the date was recorded as DG 1, the male was returned to its home cage and the female placed into a new cage with clean bedding. If no plug was observed, the male and female remained together until a vaginal plug was observed. If mating was unsuccessful for more than 5 days, the female was eliminated from the study and was euthanized.
Once a vaginal plug was confirmed, dams were weighed 3 to 4 days per week at a rate of approximately every other day. Daily monitoring of water intake continued throughout the gestation period. As the expected parturition dates neared, animals were observed two to three times each day for the initiation of parturition. PPD 1 was considered as the day when the first pup was observed in the cage. Dams were not weighed on PPD 1, but were weighed 3 to 4 days per week until PPD 10 beginning on either PPD 2 or 3. All pups within a litter were weighed on PPD 5, when the litters were culled to eight pups of four males and four females, or as close as possible to this combination. Pups and dams from any litters with less than eight pups were eliminated from the study and euthanatized. During PPDs 5 to 10, pups’ tails were tattooed in a dot pattern which separated males and females and identified individual pups within a litter.
In-Life Observations
The P generation female rats were observed for viability at least twice each day of the study. The rats were examined daily during the exposure period for clinical evidence of the effects of the test substance, abortions, premature deliveries, and deaths (OECD, 2000). Body weights were recorded twice during acclimation, daily throughout the exposure period and at sacrifice.
DL 1 was defined as the day of birth and was also the first day on which all pups in a litter were individually weighed. The duration of gestation, litter size, and pup viability were recorded at birth. Pup clinical observations and viability were recorded daily and pup weights were recorded PPDs 1, 5, 8, 10, 14, and 22. Maternal behavior was evaluated on DLs 1, 5, 8, 14, and 22. Feed and water consumption values were recorded daily during the exposure and postexposure periods. Feed and water consumption values were not tabulated after DL 14, when it was expected that pups would begin to consume the maternal feed and water.
On GD 21, P generation female rats were sacrificed and blood and tissue samples were collected as described above. A gross necropsy of the thoracic, abdominal, and pelvic viscera was performed. The number of corpora lutea in each ovary was recorded. The uterus of each rat was excised and examined for pregnancy, number and distribution of implantations, live and dead fetuses, and early and late resorptions.
The brain from one male and one female pup per litter were selected for histopathological and morphometric evaluation. One set of male and female pups was selected for evaluation from pups sacrificed on PPD 5, a second set was selected for evaluation from pups sacrificed on PPD 10, and a third set was selected for evaluation from pups sacrificed on PPD 22. Brain weights of pups sacrificed on PPD 22 were recorded post fixation. On PPD 5, litters were standardized to eight pups each (four males and four females, when possible).
Motor Activity
Opto-Varimex activity meters from Columbus Instruments, Columbus, OH, were 17 inch × 17 inch Plexiglas open fields with infrared photocells placed 2.4 cm apart along the perimeter of the fields. There were two different levels of photocells to detect both horizontal and vertical movements, as well as differentiate small (stereotypic) from large movements. Nine measures of motor activity were automatically recorded: (1) frequency of ambulatory movements; (2) time of ambulatory movements; (3) frequency of stereotypic movements; (4) time of stereotypic movements; (5) frequency of movements in the horizontal plane; (6) distance traveled in the horizontal plane; (7) frequency of rears; (8) total number of horizontal movements made while in the rearing position (vertical plane movements); and (9) time spent resting.
On PPD 14, one male and one female pup were randomly selected from each litter to be used in motor activity testing. The same animals were tested on PPDs 14, 18, and 22. On each test day, pups were placed in individual transport cages, similar to their home cages and lined with fresh bedding, for moving the animals to the testing room. Pups were left in the transport cages in the test room for 5 to 7 min to habituate to the low red lighting (25-W bulbs placed above the testing boxes) and white noise (70 dB). Pups were individually tested for 90 min in the Opto-Varimex activity meters. Each pup was placed directly in the middle of the open field and left undisturbed for 90 min, after which time they were returned to the transport cages and returned to their home cages. Between each test, the open fields were washed down with a diluted Nolvasan solution to remove urine, fecal boli, and other olfactory cues. All testing was completed between the hours of 0830 and 1430. Pups and dams were sacrificed following the final test session on PPD 22.
Neuropathology
F1 Generation Day 10 Postpartum Rats
One hundred sixty (80 male and 80 female) pups were assigned on PPD 10 for fixed brain weights. Of these pups, 14 to 16 rat brains per sex per exposure group were subsequently selected for histological examination. On PPD 10, pups selected for histological examination were sacrificed by carbon dioxide asphyxiation, the heads removed, and immersed in neutral-buffered 10% formalin. The heads were shipped to the pathologist, who removed and weighed the brains. All remaining pups were sacrificed and necropsied for gross lesions on PPD 12.
The fixed brains were removed from the cranial vault. Without knowledge of the exposure group, two anterior to posterior linear measurements (in millimeters) were taken with a Vernier caliper: (1) the cerebrum from the anterior to posterior pole, exclusive of the olfactory bulbs; and (2) the cerebellum extending from the anterior edge of the cerebellar cortex to the posterior pole (posterior edge of the cerebellar vermis). This measurement was made on a diagonal.
The brains were then weighed and divided by six cuts into coronal slices of approximately 2 mm in thickness: (1) half-way between the ventral base of the olfactory bulbs and the optic chiasm; (2) just anterior to the optic chiasm; (3) just anterior to the infundibulum; (4) through the midbrain at the level of the mammillary body; (5) through the cerebellum just anterior to its midpoint; and (6) through the anterior portion of the medulla. Cuts 1 through 4 were made from the ventral aspect of the brain, whereas cuts 5 and 6 were made from the dorsal aspect of the brain. The olfactory bulbs were also processed, resulting in a total of eight slices for each brain.
Brain slices were embedded in paraffin, step-sectioned (approximately 60
Thickness of the frontal cortex. This measurement, taken bilaterally, was of the dorsal portion of the cerebral cortex within the coronal section passing through the region of the optic chiasm and anterior commissure (Figure 1).
Thickness of the parietal cortex. This measurement, taken bilaterally, was of the dorsolateralcerebral cortex within the coronal section, passing through the region of the optic chiasm (Figure 1).
Diagonal (maximal) dimension, taken bilaterally, of the striatum and underlying globus pallidus, but not including the underlying nucleus accumbens (Figure 1).
Thickness of the corpus callosum, taken bilaterally, within the coronal section passing through the region of the optic chiasm (Figure 1). This measurement was taken at the level of the external granular layer of the overlying cingulate gyrus.
Thickness of the hippocampal gyrus. This measurement, taken bilaterally, was performed on the dorsal portion of the hippocampus within the section taken at the level of the infundibulum (Figure 2).
Thickness of the dentate gyrus of the hippocampus. This measurement, taken bilaterally, was performed on the dorsal portion of the hippocampus within the section taken at the level of the infundibulum (Figure 2).
Thickness of the CA1 portion of the hippocampus. This measurement, taken bilaterally, was performed on the dorsal portion of the hippocampus within the section taken at the level of the infundibulum (Figure 2).
Thickness of the CA3 portion of the hippocampus. This measurement, taken bilaterally, was of the distance between the alveus and the dorsal blade of the dentate (Figure 2).
Maximum height of the cerebellum at the level of the deep cerebellar nuclei, extending from the roof of the fourth ventricle to the dorsal surface (Figures 3, 4).
Thickness of the external granular (germinal) layer of the cerebellum. Six areas were measured over the dorsum of the cerebellum because the thickness of this layer varies considerably from region to region. The mean value of the six measures was recorded as one measurement.
Each measured section was digitally scanned using a Path-Scan Enabler and a Polaroid SprintScan 35 film scanner at a resolution of 1350 or 2700 dpi and a prescanned calibrated frame to standardize the image size. An attempt was made to specify the level of the first two measured sections according to
F1 Generation Day 22 Postpartum Rats
One hundred sixty (80 male and 80 female) pups were assigned on PPD 22 for fixed brain weights. On PPD 22, pups selected for histological examination were sacrificed by carbon dioxide asphyxiation and the heads removed and immersed in neutral-buffered 10% formalin. The heads were shipped to the Experimental Pathology Labs, Inc. (Herndon, Virginia), where the brains were removed, weighed, trimmed, processed, and sectioned. The slides were then sent to Consultants in Veterinary Pathology, Inc. (CVP), Monroeville, Pennsylvania, for evaluation and morphometric measurements. All remaining pups were sacrificed and necropsied for gross lesions on PPD 12. Between 15 and 16 brains per sex per exposure group were subsequently histologically examined. The methodologies were the same as those performed on the PPD 10 rats, except that the rotary microtome was set at 5
Twenty-one linear morphometric measurements (2 gross and 19 microscopic) were taken from each of the PPD 22 rat brains (18 measurements actually represent bilateral brain regions). Microscopic morphometric measurements, listed below, were made using a calibrated ocular micrometer.
Thickness of the frontal cortex. This measurement, taken bilaterally, was of the dorsal portion of the cerebral cortex within the coronal section passing through the region of the optic chiasm (Figure 5). Thickness of the parietal cortex. This measurement, taken bilaterally, was of the dorsolateral the cerebral cortex within the coronal section passing through the region of the optic chiasm (Figure 5). Diagonal (maximal) dimension, taken bilaterally, of the striatum and underlying globus pallidus, but not including the underlying nucleus accumbens (Figure 5). Thickness of the corpus callosum, taken bilaterally, within the coronal section passing through the region of the optic chiasm (Figure 5). This measurement was taken at the level of the external granular layer of the overlying cingulate gygus. Thickness of the corpus callosum, taken bilaterally, within the coronal section passing through the infundibulum (Figure 6). This measurement was also taken at the level of the external granular layer of the overlying retrosplenial cortex. Thickness of the hippocampal gyrus. This measurement, taken bilaterally, was performed on the dorsal portion of the hippocampus within the section taken at the level of the infundibulum (Figure 6). Thickness of the dentate gyrus of the hippocampus. This measurement, taken bilaterally, was performed on the dorsal portion of the hippocampus within the section taken at the level of the infundibulum (Figure 6). Thickness of the CA1 portion of the hippocampus. This measurement, taken bilaterally, was performed on the dorsal portion of the hippocampus within the section taken at the level of the infundibulum (Figure 6). Thickness of the CA3 portion of the hippocampus. This measurement, taken bilaterally, was of the distance between the alveus and the dorsal blade of the dentate (Figure 6). Maximum height of the cerebellum at the level of the deep cerebellar nuclei, extending from the roof of the fourth ventricle to the dorsal surface (Figure 7).
Each measured section was digitally scanned using the same equipment and methodologies as those performed on the PPD 10 rats.
Statistical Analyses
The statistical methods for the in-life and reproductive data were as follows. Clinical observations and other proportion data were analyzed using the variance test for homogeneity of the binomial distribution (Sokal and Rohlf 1969a). Continuous data (e.g., maternal body weights, body weight changes, feed consumption data, organ weights, duration of gestation, litter averages for pup body weights, percent male pups, pup viability, and cumulative survival) were analyzed using Bartlett’s test of homogeneity of variances (Sokal and Rohlf 1969a) and the analysis of variance (Snedecor and Cochran 1967), when appropriate (i.e., Bartlett’s test was not significant [
The statistical methods for the quantitive morphometric data were conducted by Syracuse Research Corporation, Syracuse, NY, using SAS Version 8 software. The analyses consisted of a Bartlett’s test of homogeneity of variances (tested at
The data for the motor activity were analyzed separately for each of the nine measures of motor activity using a univariate repeated measures ANOVA. The between-subjects variable was AP dosage, with five levels. The three within-subjects variables were sex (two levels), age (three levels), and time block (nine levels [nine blocks of 10 min each]). Due to violation of the sphericity assumption, the more conservative Greenhouse-Geisser test was employed. The fiducial limit was set at
RESULTS
Consumed Dosages
The mean consumed dosages for the female rats at the Pennsylvania site during the precohabitation, gestation and lactation exposure periods are shown in Table 1a. The average actual consumed daily dosages (mg/kg-day) for the female rats for the precohabitation exposure period (days 1 to 14) were 0.00, 0.01, 0.09, 0.77, and 23.80 mg/kg-day for the 0 (carrier), 0.01, 0.1, 1.0, and 30.0 mg/kg-day target dosage groups, respectively. The average actual consumed daily dosages for the female rats for the gestation exposure period (DGs 0 to 21) were 0.00, 0.01, 0.09, 0.97, and 28.34 mg/kg-day for the 0 (carrier), 0.01, 0.1, 1.0, and 30.0 mg/kg-day target dosage groups, respectively. The average actual consumed daily dosages for the female rats for the lactation exposure period (DLs 1 to 10) were 0.00, 0.01, 0.12, 1.26, and 38.32 mg/kg-day for the 0 (carrier), 0.01, 0.1, 1.0 and 30.0 mg/kg-day target dosage groups, respectively.
The mean consumed dosages for the female rats during the entire exposure periods at the Ohio site are shown in Table 1b. The average consumed daily dosages (mg/kg-day) for the female rats were 0.00, 0.13, 1.35, 3.71, and 12.39 mg/kg-day for the 0 (carrier), 0.1, 0.1, 3.0, and 10.0 mg/kg-day target dosage groups, respectively.
In-Life
There were no deaths, adverse clinical observations, or necropsy findings during the premating, gestational, and/or lactation periods for the portion of the research conducted in Pennsylvania that was considered exposure related. Average body weights and body weight changes for female rats were comparable among the five exposure groups through the precohabitation, gestation, and/or lactation periods (Figure 8). Average maternal absolute and relative water and feed consumption values for female rats during the precohabitation, gestation, and/or lactation periods were comparable among the five exposure groups (data not shown). Cesarean-sectioning observations were based on 16 pregnant dams on DG 21 in the four respective dosage groups. Dosages up to 30.0 mg/kg-day did not affect any Cesarean-sectioning or litter parameters. There were no statistically significant differences for average numbers of corpora lutea, implantations, preimplantation loss, litter size, early or late resorptions, sex ratio, or fetal weights among the five exposure groups. Fifteen or 16 pregnant dams that delivered were selected in each of the 0 (carrier), 0.01, 0.1, 1.0, and 30.0 mg/kg-day target dosage groups, respectively. Natural delivery observations were unaffected by targeted dosages of the test substance as high as 30.0 mg/kg-day (see accompanying article by York et al.). There were no statistical differences for gestation length, implants, litter size, stillborn, or pup viability among the five exposure groups. Pup weights were significantly increased in the 0.01, 1.0, and 30.0 mg/kg-day exposure group the second and/or third week of lactation. These increases were considered related to the decreased weight of the control pups and not to the test substance for the exposure groups. All clinical and necropsy observations in the F1 generation pups were considered unrelated to exposure to the test substance.
There were no deaths, adverse clinical observations, or necropsy findings during the course of the study conducted in Ohio that were considered exposure related. Mean body weights and body weight changes for female rats were comparable among the five exposure groups and did not differ significantly (data not shown). Due to non–exposure-related attrition, statistical analyses were completed for 84 litters of the original 110 dams: 15 control litters, 18 at 0.1 mg/kg/day, 19 at 1.0 mg/kg/day, 17 at 3.0 mg/kg/day, and 15 at 10.0 mg/kg/day.
Motor Activity
No statistically significant decreases were found for the main effect of consumed dosage for any of the nine measures of motor activity, and there were no reliable interactions related to exposure. This suggests minimal effect of ammonium perchlorate on the measures of rat pup general locomotor activity. However, a general pattern in the results shows that, in several instances, there was a notable divergence in activity between the control group versus dosed groups, and this difference emerged late in the 90-min testing sessions (Figures 9 to 11). Patterns in the present data as well as the previous study (York 1998; York et al. 2004) suggest that exposed pups may have a slightly slower rate of habituation, and thus maintain a higher level of activity as compared to untreated pups.
As expected, there was a main effect for age in all nine measures of motor activity. In most cases, there was an increase in locomotion from PPDs 14 to 18, and a slight reduction from PPDs 18 to 22. The only measures that deviated from this pattern were rears and movement in the vertical plane, where a consistent increase was observed with increasing age. A reliable main effect for time block was also found for all nine dependent variables due to an overall decrease in behavior from start to finish of each test session. The time-dependent reduction in motor activity was less evident on PPDs 14 and 18, than on PPD 22, as indicated in the significant two-way, Age × Time block, interaction (Figure 11). The interaction was reliable for all motor activity measures; however, the measures of rears and vertical plane movements were not consistent with this general pattern. Rather, for these two measures, decreases from the start to finish of the test sessions were found on PPDs 14 and 22, but on PPD 18 the number of rears was the same at the end as at the beginning of the 90 minutes, and vertical plane movements increased from the beginning to the end of the session.
The three-way interaction of Sex × Age × Time block was significant for some measures, specifically, time ambulatory, stereotypic bursts, stereotypic time, horizontal movements, and time resting. Overall, the primary difference between the females and males is found on PPD 14 during the time blocks near the midpoint of the 90-min test session. For the measures of time ambulatory, stereotypic bursts, horizontal movements, and time resting, the females demonstrated a slight decrease in activity whereas the males demonstrated a slight increase.
For the measure of vertical plane movements, there was a significant Sex × Time block interaction. The effect was due to a greater decrease in these movements during the earlier half of the test session in females as compared to the males. However, the effect was not reliable for any of the other dependent measures.
Brain Weights, Neuropathology, and Morphometry
Detailed microscopic examination of multiple coronal sections from PPDs 10 and 22 rat pup brains in each of the 0.0 and 30.0 mg/kg-day exposure groups did not indicate any evidence of exposure-related neuropathologic alterations or of microscopic developmental anomalies. The brains from the PPD 10 pups were characterized by active cellular migration and cell death (i.e., physiologic cell death or apoptosis) as well as ventricular remodeling.
The PPD 10 rat pup brain weights and morphometric measurements are presented in Tables 2a and 2b. The male pup brain weights in the 30 mg/kg-day exposure group were slightly increased (3.8%) over the carrier-control exposure group. For the male rats, there was a trend for increased linear dimensions in a number of brain regions, especially for the posterior level of the corpus callosum and, to a lesser extent, for certain hippocampal and cortical thickness measures. For the female rats, the pattern was reversed. The female data were more variable between groups, with some dimensions being of lesser value and some of greater value than for the female control group. In the PPD 10 male pups, the right and left corpus callosum measurements were significantly greater only in the middle two dose groups (0.1 and 1.0 mg/kg-day), and not the high-dose group (30.0 mg/kg-day). The left CA3 was significantly greater only at the high dose. The remaining measurements that were statistically significant were only significant in the 1.0 mg/kg-day group and no other dosage groups. In the PPD 10 female pups, the right and left CA1 were significantly greater only in the mid-dose two groups, and not in the low- and high-dosage groups. The remaining measurements that were statistically significant were only significant in the 1.0 mg/kg-day group (except that the external granular layer of the cerebellum was also significantly thicker in the 0.01 mg/kg-day dosage group) and no other dose groups. No effect was observed on the thickness/height of the cerebellum at PPD 10.
The pattern of intergroup differences observed in the morphometric parameters for the PPD 22 rats (Tables 3a and 3b) are similar to those encountered with the PPD 10 rats present on this study, even though the slides of brain tissue from the rats of these two ages were produced at different histology laboratories. At both age points, the brains of male rats tended to have a number of thicker neuroanatomic regions than similar regions in control group brains, whereas the reverse trend was present for the females. These data suggest not only a treatment-related effect on brain development, but that there may also be some degree of sexual dimorphism regarding response of rats to the test substance (Table 4).
DISCUSSION
Data from previous research suggests that hypothyroidism interferes with normal brain development, including changes in the brain areas critical for the integrity of motor coordination (Pasquini and Adamo 1994; Chan and Kilby 2000). If there are abnormalities during maturation of locomotor control systems in the brain, it is likely that these would be revealed in tests of neurobehavior related to those systems. In the present investigation, the test of open field motor activity was used to evaluate pups from dams treated with different doses of AP in their drinking water. Overall activity of an animal in an open field can be used to assess the integrity of brain systems related to gross motor movement, general exploratory activity, and habituation to a new environment. The open field measure can quantify animal activity ranging from totally unresponsive to hyperactive, and thus is ideal for assessing both increases and decreases in overall locomotor activity.
The behavioral testing results suggest that a pregnant dam’s exposure to AP does not reliably affect the development of gross motor movements in the pups. The lack of effect on this neural system was demonstrated at three postweaning ages, where no differences were found between any of the dose groups for a variety of motor activity variables. Although previous research suggests that AP exposure may cause abnormalities in cerebellar development that could be manifested in changes of locomotor behavior, the current evidence does not support such a prediction. However, a pattern did emerge, suggesting there may be a slightly slower habituation response to the testing environment related to previous AP exposure (i.e., resulting in a higher level of activity as compared to untreated pups). A similar pattern was reported in the results from a closely related investigation (York 1998; York et al. 2004). In such cases, an exposure related effects would be due less to the integrity of the neural substrates specific for motor activity, and more likely related to general brain systems for behavioral inhibition or those affecting memory. An appropriate follow-up to the present research would be to employ neurobehavioral tasks to specifically investigate habituation to novel stimuli or environments. For instance, future studies may use new animals for testing at each of the different ages in order to maximize the novelty of the testing situation. Such methodology may increase the sensitivity of the open-field locomotor activity test to changes in the rats’ patterns of habituation, as well as investigate the integrity of the motor system in response to AP exposure.
The mean values for the linear dimensions of a number of brain regions for both PPDs 10 and 22, male and female rat pups, in this study were found to be either significantly greater or lesser for certain AP-treated groups, than for the comparable control groups. However, a sexually dimorphic pattern was evident for a number of the treatment-related measurement differences. For example, AP treatment generally had a trophic effect on the male rat brains, with a number of brain regions in certain AP-treated groups being of significantly greater dimension than in the male rat control groups. For the female rats, on the other hand, the means of many measures were shorter in AP-treated groups. In spite of this, there were no statistically significant intergroup differences in brain weights. For both the PPDs 10 and 22 male rats, significantly greater thicknesses/widths were seen for one or more male AP-treated groups (except for the 0.01 mg/kg-day group) for at least the following measures: cerebral cortex (frontal and parietal), corpus callosum, and hippocampus. For the PPD 22 male and female rats, the heights of the cerebellum were also increased significantly in some AP-treated groups. For the male rats, the treatment-related differences were most prominent for the 1.0 mg/kg-day group; this group showing a greater effect than the 30.0 mg/kg-day group. However, some measures were also significantly greater (than male control group values) for the 0.1 and 0.01 mg/kg groups. These findings stand in contrast to most of the published literature on hypothyroidism in rats (showing decreased thicknesses of various brain structures in association with hypothyroidism) (e.g., Berbel et al. 2001).
The most consistent treatment-related effect in the PPDs 10 22 male rats was the increased thickness of the corpus callosum. For the PPD 10 male rats, the corpus callosum was significantly thicker (versus control group values) for both the 0.1 and 1.0 mg/kg-day exposure groups. For the PPD 22 male rats, the body of the corpus callosum was significantly thicker for three treatment groups (i.e., 0.01, 0.1, and 1.0 mg/kg/day). Intergroup differences in mean corpus callosal thicknesses were on the order of approximately 20% to 30% (greater for AP-treated rats). However, there was no evidence of a dose-response. Although the mechanism for a treatment-related increase in the thickness of the corpus callosum could not be determined, the young ages of the rats in this study (PPDs 10 and 22) indicate that the degree of myelination is not likely to have been a factor. That is, the corpus callosum is one of the last tracts to undergo myelination in the rat brain (Hamano et al. 1998; Nunez et al. 2000). On the other hand, it is possibly that a delay in the onset of programmed cell death (which is at least partially under thyroid hormone control) could result in increased numbers of cortical projecting neurons and a decrease in the attrition of axons within the corpus callosum. It is generally accepted that some neurons projecting across the corpus callosum during early development subsequently undergo programmed cell death, thus resulting in the disappearance of their axons from the corpus callosum (Ding and Elberger 2001). A treatment-related delay in the onset of programmed cell death could potentially also explain the relative lack of a dose-response (e.g., if programmed cell death is either switched on or off rather than being variably modulated). On the other hand, hypothyroidism has not been demonstrated to affect the numbers of axons in the corpus callosum—or, at least, not until timepoints later than those in this study (Berbel et al. 1994; Guadano-Ferraz et al. 1994). A sexually dimorphic response has been reported in the development of the splenium of the rat corpus callosum (Kim and Juraska 1997). However, because developmental differences exist between the various levels of the corpus callosum, exploring the mechanism of perchlorate treatment-related corpus callosum development will undoubtedly require the examination of sagittal sections of this structure (measuring for area and overall configuration) at multiple postnatal developmental time points.
For both the male and female PPD 22 (but not PPD 10) rats, the means of the full thickness of the cerebellum were found to be thicker for all of the test substance–exposed groups. These increases were statistically significant for the male rats in the 0.01, 0.1, and 1.0 mg/kg-day test substance–treated groups, as well as for female rats in the 0.01 and 1.0 mg/kg-day exposure groups. As with the measures previously discussed, there was a flat dose-response curve and no effect of treatment in the high-exposure group. Previous research by other investigators has suggested that the cerebellum is exquisitely sensitive to the deleterious consequences of a number of toxic chemicals (Fonnum and Lock 2000). Furthermore, development of the cerebellum is also vulnerable to the effects of early deficiencies in thyroid hormones. For example, reduced myelin formation has been reported in the cerebellum of hypothyroid rat pups as compared to controls (McIntosh et al. 1981; Pasquini and Adamo 1994). Also, reduced cerebellar weight (Walker et al. 1989), and smaller cell size of cerebellar neurons (McIntosh et al. 1981) have been reported for hypothyroid rat pups. The smaller-sized cerebellar neurons (McIntosh et al. 1981) have been reported for hypothyroid rat pups. The smaller cell size could be indicative of delayed or disrupted cell differentiation, an effect further evidenced in elevated neural density and smaller average neuron size in pups from dams fed a severely iodine deficient diet (Li et al. 1986). These changes suggest that early exposure to perchlorate, and the effects it has on thyroid hormones, may result in abnormal cerebellar development. Given the critical role of the cerebellum in general motor activity and coordination (Middleton and Strick 2000; Armstrong, Apps, and Marple-Horvat 1997), an early insult at this level may be predicted to manifest itself in atypical patterns of spontaneous locomotor responding. Yet, no statistically significant decreases were found for the main effect of exposure for any of the nine measures of motor activity, and there were no reliable interactions related to drug dose.
Studies on the effects of hypothyroidism on brain development have generally shown decreased sizes in selected brain structures, whereas this study on ammonium perchlorate has revealed a sexually dimorphic response, with some brain regions being larger in perchlorate-treated male rats than in comparable controls. Although the relatively flat dose responses seen in this study might suggest that the control groups were not representative, these data also suggest the possibility that there may be a sexually dimorphic response in the control of programmed cell death by thyroid hormone. The lack of any clear and consistent dose-response relationships, apparent systematic differences in the plane of sectioning of the brain, and doubts the biologic plausibility of the changes observed raise questions and concerns on any of the brain development findings. Additional studies employing more sophisticated morphometric techniques will be required to determine the mechanisms behind any perchlorate treatment-related effects.
Footnotes
Figures and Tables
Acknowledgements
The authors gratefully acknowledge the services provided by Latha Narayanan, Geo-Centers, Inc. at AFRL/HE for perchlorate analyses and William Stiteler at Syracruse Research Corporation, Syracuse, NY, for providing statistical support for the quantitative morphometric data. The authors also thank Rosalyn Garman for her input in the histotechnology component of this study. Funding for this research was by the Perchlorate Study Group, Michael Girard, Chairman.