Differential and Non-Uniform Tissue and Brain Distribution of Two Distinct 14C-Hexachlorobiphenyls in Weanling Rats

Shakil A. Saghir*,1, Larry G. Hansen*, Kenneth R. Holmes* and Prasada R. S. Kodavanti{dagger}

* Department of Veterinary Biosciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802; and {dagger} Cellular and Molecular Toxicology Branch, Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711

Received July 19, 1999; accepted November 2, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Excretion and tissue retention of a coplanar and a non-coplanar hexachlorobiphenyl (HxCB) were determined 48 h after a single intraperitoneal (ip) dose of 8 mg/kg radiolabeled [14C]-HxCBs to weanling male and female Long-Evans rats. The objective was to understand the involvement of initial target organs of chlorobiphenyl (CB) accumulation following acute exposure in immature animals. During the short interval, both HxCBs remained sequestered predominantly in mesenteric fat (compared to subcutaneous fat) and less than 1% of the doses were excreted. Excretion was 4- to 8-fold lower than adult rats. Coplanar CB 169 (3,3',4,4',5,5'-HxCB) did not accumulate appreciably in the brain, but was retained at 3-fold higher levels in the liver than was non-coplanar CB 153 (2,2',4,4',5,5'-HxCB). Accumulation of 14C-CB 153 in brains was 4- to 9-fold higher than that of 14C-CB 169 and was adequate to detect non-uniform distribution in serial cryostat sections by phosphor imaging autoradiography. The autoradiographs showed a higher CB 153-derived radioactivity associated with fiber tracts throughout the brain. Specifically, the corpus callosum, internal and external capsules, medial lemniscus, tegmentum of the mesencephalon and metencephalon, and cerebellar peduncles showed significantly higher 14C-CB 153 than the other structures. The 14C-CB 153 was not found in the ventricular system and vascular spaces. These results suggest for the first time that an ortho-substituted PCB congener accumulated preferentially in brain in a structure-specific manner when compared to a non-ortho-substituted PCB congener.

Key Words: brain; polychlorinated biphenyl (PCB); neurotoxicity; structure-specific brain accumulation; 2,2',4,4',5,5'-hexachlorobiphenyl; 3,3',4,4',5,5'-hexachlorobiphenyl; PCB distribution; PCB excretion.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polychlorinated biphenyls (PCBs) are ubiquitous global contaminants causing a wide spectrum of biological effects. The most potent congeners are those which are coplanar and resemble 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); on the other hand, the most prevalent are those with ortho chlorine substitutions favoring the non-coplanar configuration (Hansen, 1994Go, 1998Go; Safe, 1994Go). The ortho-chlorinated (non-coplanar) congeners are detected in higher quantities in all environmental (soil, water, and air) and biological (human blood, human milk, dairy and meat products) samples (Hansen, 1998Go; WHO, 1993). Both types of congeners as well as mixtures have been reported to cause learning and behavioral abnormalities following in utero and/or neonatal exposure (Huisman et al., 1995Go; Jacobson and Jacobson, 1996Go; Schantz, 1996Go; Schantz et al., 1995Go; Seegal and Schantz, 1994Go; Tilson et al., 1990Go); however, non-coplanar congeners act through direct neurochemical mechanisms of toxicity that appear to be unrelated to the Ah receptor (Kodavanti et al., 1995Go, 1996Go, 1998Go; Kodavanti and Tilson, 1997Go; Kodavanti and Ward, 1998Go; Seegal et al., 1990Go; Schantz, 1996Go; Shain et al., 1991Go; Tilson and Kodavanti, 1998Go; Wong and Pessah, 1996Go).

Non-coplanar PCB congeners have been reported to preferentially accumulate in rat brain, while coplanar congeners appear to be excluded (Sparling and Safe, 1980Go). Accumulation in brain has also been reported to be unequally regionalized, based on chemical analysis of grossly dissected regions (Kodavanti et al., 1998Go; Ness et al., 1994Go; Seegal et al., 1990Go, 1991Go). Structure-specific brain distribution could neither be demonstrated by these techniques nor even by autoradiography following exposure to 14C-tetrachlorobiphenyls (Ness et al., 1994Go).

Although adverse developmental outcomes have been associated with PCBs in humans, wildlife, and laboratory animals, adult steady-state exposures appear to be of little consequence. Immature rats were used in studies to address these concerns regarding developmental effects and to better define disposition in the immature animal. Rat development, especially of the nervous system and reproductive tract (including hormone receptors and sexual differentiation) continues through the prepubertal period (Dohler, 1986Go; Hoar and Monie, 1981Go; Rodier, 1980Go). Drug-metabolizing enzymes (both phase 1 and phase 2) also develop and differentiate postnatally (Lucier, 1978Go; Klinger et al., 1981Go). In addition, immature animals have less body fat for the sequestration of persistent lipophilic chemicals such as PCBs. Short-term exposures better define the initial actions of neuroendocrine-active compounds for structure-activity relationships. The disposition also mimics the food chain exposures to pulsatic, higher-than-ambient levels through, e.g., a fish meal. These sporadic PCB exposures appear to be accommodated by adults, but may be detrimental to developing animals because of more vulnerable targets and a lower capacity for elimination and/or sequestration (Hansen, 1998Go).

The objective of this study was to investigate differences in the short-term excretion and distribution, especially localization within brain, of two structurally distinct hexachlorobiphenyls (HxCBs) in weanling (prepubertal) male and female rats. Since initial actions of developmental toxicants cannot be determined following prolonged dosing, and individual doses can not be determined accurately from in utero or lactational exposures, weanling rats were used to approximate the acute disposition of pulsatic PCBs in developing animals. One of the most important non-coplanar PCB congeners, CB 153 (2,2',4,4',5,5'-HxCB), is found in all environmental samples and accounts for 15–30% of total PCB in most human samples (Jensen and Sundstrom, 1974Go; Hansen, 1998Go; WHO, 1993). The distribution of 14C-CB 153 was compared to coplanar 14C-CB 169 (3,3',4,4',5,5'-HxCB) following a single intraperitoneal (ip) injection in the weanling rats. These two HxCBs differ in affinities for the Ah-receptor, enzyme induction potencies and patterns, susceptibility to metabolism, effects on second messenger systems, and effects on cellular dopamine levels (Hansen, 1998Go; Kodavanti and Tilson, 1997Go; Safe, 1994Go; Shain et al., 1991Go). The localization of these radiolabeled [14C]-HxCBs within the brains was determined by phosphor imaging using lyophilized cryo-sections. This technique is 10 times more sensitive than traditional autoradiography and, for the first time, demonstrated non-uniform distribution of 14C-derived PCBs and/or their metabolite(s) in different anatomical regions of the brain. This approach can be used, after further improvements, to direct more detailed investigations in order to find specific distribution of compounds within the brain to better understand the related neurotoxicity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Uniformly-radiolabeled CB 153 (14C-2,2',4,4',5,5'-HxCB) (specific activity 12.6 mCi/mmol; >99% chemically pure) and CB 169 (14C-3,3',4,4',5,5'-HxCB) (specific activity of 12.3 mCi/mmol; >99% chemically pure) were purchased from Sigma Chemicals (St. Louis, MO). Radiochemical purity of CB 153 was 98% and of CB 169 was 100%. Non-radioactive CB 153 (>99% purity) was purchased from Ultra Scientific (North Kingstown, RI). Dosing solutions of the required concentrations were prepared in corn oil.

Animals (care and dosing).
Female Long-Evans rats nursing a litter of 16-day old pups (4 males and 4 females) were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Animals were housed in polycarbonate cages with corncob bedding and stainless steel wire tops under standard conditions (23 ± 2°C, 55% relative humidity, 12-h light/dark cycle). Rats were provided with ground purified AIN 93 G diet fortified with 2.0 ppm iodine (Harlan-Teklad, Madison, WI) and water ad libitum. Pups were weaned at the end of day 20 and placed in individual metabolism cages. For each HxCB, 3 male and 3 female pups from the same litter were designated to receive the radioactive HxCBs and the 4th received the control solution. Following 12 h of acclimatization, each animal (weighing between 40–52 g) was dosed intraperitoneally with 0.1 ml of either 14C-CB 153 or 14C-CB 169 (8 mg/kg; 12.5 µCi/rat) in corn oil on postnatal day 21. Control animals received an equal amount of corn oil. Additional control animals were introduced from 2 additional litters, used to confirm survival following 8 mg/kg CB 169 dosing. In the CB 153 group, non-radiolabeled CB 153 was also administered simultaneously to obtain the total dose of 8 mg/kg. Thus, there were 3 males and 3 females from single litters in each HxCB group. For organ weight and response comparison, 2 additional control males and females were used (making the total of 3 male and 3 female control animals), but their tissues were not extracted for measurement of radioactivity.

In the CB 169 group, only radioactive chemical was used to make up the dose. Higher radiolabeled CB 169 was thought to be needed for an appreciable brain accumulation since coplanar PCBs have been reported to have less affinity for brain (Kodavanti et al., 1998Go; Ness et al., 1994Go; Saghir et al., 1999Go; Sparling and Safe, 1980Go).

Sample collection.
Urine and feces were collected separately every 2 h up to 12 h and then every 6 h up to 48 h. The animals were weighed and euthanized by decapitation 48 h after dosing (at 23 days-of-age). Blood was collected from cervical stumps. Brains were carefully removed from the skull immediately (within 2–3 min) after decapitation and frozen on dry ice. Each brain was placed vertically on the brain stem, covered with powdered dry ice to hasten freezing and maintain anatomical features, and stored at –70°C until sectioning. The area around the site of injection (~1 cm2) was excised and collected to monitor sequestration of doses. Radioactivity found in the excised skin and associated subcutaneous fat around the injection site was subtracted from the total injected radioactivity. Urinary bladders were manually emptied and the collected liquids were added to the corresponding urine samples. Various organs (liver, kidney, spleen, testis/ovary, thymus, mesenteric and subcutaneous fat, and brown fat) were removed from each animal by gross dissection within 5 min after decapitation. Dissecting instruments were washed in acetone between tissues, to avoid cross-contamination. All collected samples, with the exception of brain, were stored at –10°C immediately after collection, until further analysis could be carried out. Since it was not possible to reliably retrieve 100% of body fat, samples of specific fat depots were considered to be representative of the entire depot.

Radioactivity in the urine was quantitated directly by taking a 10% aliquot by weight for liquid scintillation counting (Packard Tri-Carb 1900 TR scintillation spectrophotometer, Packard Instrument Co., Downers Grove, IL) in ScintiVerse BD (Fisher Scientific, Fairlawn, NJ) scintillation cocktail. Feces were thoroughly mixed before taking aliquots. The frozen brains were serially sectioned, from the olfactory lobe to the brain stem, and two sections every 300–350 µm apart were saved for phosphor imaging. All shavings and unused sections from each brain were pooled, homogenized and total radioactivity was determined in an aliquot of the homogenate. Total radioactivity in other tissues was quantitated in weighed aliquots (~0.2 g). Samples were taken from at least 3 different regions of each tissue and digested overnight in 1–2 ml Soluene-350 tissue solubilizer (Packard, Meriden, CT) at 50°C. Colored samples were decolorized by adding a maximum of 0.2 ml of 30% H2O2, pH was neutralized by adding 100–200 µl of acetic acid to eliminate chemiluminescence, and radioactivity was determined in ScintiVerse II (Fisher Scientific, Fairlawn, NJ).

The scintillation counter was calibrated to correct for quenching by establishing a curve using quench standards. Rate of chemiluminescence was also monitored and samples were counted until chemiluminescence was completely decayed. The counting efficiency of the scintillation counter was >95%. Background radioactivity from a non-14C-treated rat was subtracted from each sample before analyzing for the concentration.

Brain processing.
The brains were processed for autoradiography using a phosphor imager after modifying the procedures described by Ness et al. (1994) for traditional autoradiography. Quick-frozen brains stored at –70°C were moved to –20°C for 8–10 h before sectioning and sectioned at –19°C using a Minotome Microtome Cryostat (International Equipment Co.). Transverse sections 16-µm thick were collected every 300–350 µm, beginning at the olfactory lobe and progressing through the cerebellum. Sections were thaw-mounted (thawed with finger on the opposite side of glass slide) on gelatin-coated microscope slides which had been stored at –70°C and were moved to –19°C one h before sectioning. Sections were dehydrated by lyophilization. All slides for each CB experiment were mounted, with control brains interspersed, on a cassette to be placed in contact with a MD28 phosphor storage screen (Molecular Dynamics, Sunnyvale, CA). A few tester slides were mounted on smaller screens for periodic screening to check exposure levels. The phosphor screens with brain samples were kept at –20°C. Localization of radioactivity within brain sections was determined by exposing the slides for 75 days (14C-CB 153) or 350 days (14C-CB 169) before detection of the stored radiographic images with the phosphor imager (425S PhosphorImager, Molecular Dynamics). The lower limit of detection for a one-h exposure to the phosphor imager screen is less than 2 dpm/mm2 for 14C. The sections were subsequently used to identify different brain regions. Standard histological techniques were employed: the sections were stained with Luxol Fast Blue and counter stained with Cresyl Violet (Prophet et al., 1992Go).

Statistics.
The excretion and accumulation data for 14C-HxCBs were analyzed by 2-tailed Student's t-test. Data in text, tables, and figures are given as means ± standard error.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The data presented in this paper are expressed as cumulative percents of the total administered dose excreted from the body, or percentage accumulated per gram of different organs. Total radioactivity does not differentiate between the parent CBs and their metabolites. The pattern of visual distribution of radioactivity in different anatomical regions of brain by phosphor imaging also represents 14C-derived parent HxCBs and/or their metabolite(s).

In 2 of the 3 female rats dosed with CB 169, extraordinarily high (100x higher than other rats) radioactivity was recovered from the excrement and cage washes. This indicated previous receiver contamination and/or misdelivery of the doses, so excretion by this group is not reported. Tissue distribution, however, was consistent with the other rats.

Toxic Responses
Figure 1Go compares animal growth and the liver and uterus weights between controls and the two HxCB treatments. The data are not definitive due to small n, but reflect relevant responses of the subjects. Both male and female rats, treated with either HxCB, had similar body weight gains during the 48 h after dosing and were not different from controls (13–15% in CB 153, 16–19% in CB 169 and around16% in controls). Relative liver weights were higher in both of the treatment groups; the increase was modest in CB 153-treated pups, whereas it was substantial in CB 169-treated rats. The differences between control and treatments as well as between treatments were highly significant (Fig. 1Go). The weight of the uterus was 20% lower in CB 169-treated females when compared to the CB 153-treated females. Ratios of the uterus-to-body-weight were 0.00065, 0.00061, and 0.00050 for control, CB-153, and CB-169 females, respectively. The difference between controls and CB 169 was statistically significant (p < 0.03), whereas the two HxCB treatments were not statistically different.



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FIG. 1. Comparison of the effects of a single intraperitoneal injection of 8-mg/kg CB 153 or CB 169 on growth and liver and uterus weights 48 h after dosing. Each bar represents the mean ± standard error of 3 animals. Asterisks represent statistically significant differences between control and treatment groups (* <= 0.03, ** <=0.006).

 
Urinary and Fecal Excretion
Table 1Go shows the percent-dose of the 2 HxCBs recovered in urine and feces within 48 h. Only a tiny fraction (<1%) of the administered HxCBs were eliminated from the body during the period of 48 h. Males excreted a total (urinary + fecal) of 0.62 ± 0.18% of the 14CB 153 and 0.68 ± 0.07% of the 14CB 169. In male pups, concentrations of both of the HxCBs were twice as high in urine as in feces (0.41 ± 0.15 vs. 0.21 ± 0.03 percent of CB 153 and 0.46 ± 0.03 vs. 0.22 ± 0.04 of CB 169 in urine and feces, respectively) (Table 1Go). Total excretion of CB 153 by females was 0.26 ± 0.09 percent of the administered dose and the urinary and fecal excretions were almost equal (0.12 ± 0.05 vs. 0.14 ± 0.04 in urine and feces, respectively). Excretion of CB 169 by the females could not be calculated due to contamination of the metabolism cages from prior usage and/or misdelivery of the dosing solutions (see Materials and Methods for detail). Males were twice as efficient in excreting CB 153 as females (Table 1Go).


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TABLE 1 Concentrations of Radioactivity Associated with Two 14CHxCBs Recovered in Urine and Feces of Prepubertal Male and Female Rats 48 h after a Single ip Injection
 
Tissue Distribution/Accumulation
Concentrations of the two 14C-HxCBs in different tissues of male and female pups 48 h after a single ip injection are shown in Table 2Go.


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TABLE 2 Concentrations of Radioactivity Accumulated in Different Tissues of Prepubertal Male and Female Rats 48 h after a Single ip Injection of Two 14C-HxCBs
 
Some of the intraperitoneally injected HxCBs were found trapped at the dosing sites (CB 153: male, 3.0 ± 0.5, female, 0.7 ± 0.1; CB 169: male, 0.4 ± 0.3, female, 1.8 ± 0.5 percent of the injected doses). The dosing-site sequestration of the CB 153 was higher in males, whereas females sequestered higher concentrations of the CB 169. Total injected radioactivity was corrected by subtracting these amounts found at the dosing sites prior to data analysis.

Both of the ip-injected 14C-HxCBs were mostly distributed initially to the mesenteric fat, where they were sequestered for an extended period of time; 11–15% of the injected HxCBs per gram of mesenteric fat was still present 48 h after dosing (Table 2Go). Redistribution of the HxCBs from mesenteric fat to other fat depots was apparently not complete at 48-h post-dosing. Concentrations of the HxCBs in subcutaneous fat were not equivalent to those in mesenteric fat 48 h after the dosing, and were 4–8-fold lower in 10 out of 12 rats (Table 2Go). The ratios of the 2 fat depots (mesenteric and subcutaneous) in 2 of the 14C-CB 153-treated rats were almost equal (data for the individual rats are not shown). Twenty-two percent higher concentrations of CB 169 were found in mesenteric fat in both male and female pups as compared to CB 153. However, there was no gender difference in the concentrations of either HxCB (Table 2Go). A relatively high concentration of CB 169 was also accumulated in brown fat (3–5% dose/g). The concentration of CB 153 in brown fat was not determined.

Concentrations of CB 153 in blood (serum and/or clotted fraction) were about twice as high as those of CB 169. Higher blood concentrations of both of the HxCBs were found in male rats (35–37% higher than females) (Table 2Go); however, the difference was statistically significant only for CB 169 (p = 0.015). As expected, CB 169 was found at 3-fold higher concentrations than those of CB 153 in liver (p = 0.001). There was no gender difference in the accumulation of either HxCB in liver. Both HxCBs were accumulated to a greater extent (3-fold) in the ovaries than testes. However, the difference was not statistically significant. A 2–3-fold higher concentration of CB 169 was found in kidneys when compared to CB 153 (Table 2Go). The difference in kidney accumulation of the 2 HxCBs was highly significant (p < 0.001); however, there was no statistical gender-related difference for CB 153 and the difference was moderate for CB 169 (p = 0.02). The total of 1–3% of the dose/g of spleen was too variable for any comparison (Table 2Go).

Accumulation and Non-uniform Distribution of 14C-HxCBs in Brain
A significantly higher concentration of the non-coplanar 14C-CB 153-associated radioactivity was accumulated in the brain as compared to radioactivity derived from coplanar CB 169 (Table 2Go). Concentrations of total CB 153 radioactivity averaged 9-fold higher than those from CB 169 in male brains (0.109 vs. 0.012 percent dose/g) and 4-fold higher in female brains (0.067 vs. 0.016 percent dose/g). The differences in the tendencies for these two HxCBs to penetrate and/or accumulate in rat brains were highly statistically significant (p <= 0.01) for both males and females.

The lower brain accumulation of 14C-CB 169 resulted in no detectable images, even after exposure to the screen for 350 days, compared to 75 days for the 14C-CB 153 rat brains. Images of the CB 153-treated rat brains ranged from very weak to very good. Phosphor images of each of the rat brains corresponded to the concentration of 14C-CB 153 in the brain. Images were barely differentiable from the background at the average brain concentration of 0.006 µCi/gm; clear regionalization was achieved at >=0.01 µCi/gm (data of individual rats not shown). Concentrations of radioactivity associated with 14C-CB 169 were <=0.002 µCi/gm in all of the rat brains, and thus, no phosphor image was achieved even after extended exposure (5 times longer than CB-153 brains). It appears that in order to achieve images of 14C-CB 169-treated rat brains that are differentiable from the background, more exposure time or, more probably, thicker sections were needed for the low levels of radioactivity found in these brains.

After imaging, examination of the histologically stained brain sections was performed to locate the appropriate plane of sectioning in the rat brain atlas (Paxinos and Watson, 1982Go). Although the cytology was distorted by tissue lyophilization, at 10x magnification, the histological staining, nevertheless, provided a clear distinction between aggregations of neurons and fiber tracts. The description of results was purposefully limited to the larger anatomical structures; smaller structures were avoided due to the nature of this study. Nonetheless, the autoradiographic images supported the fact that 14C-CB 153 was distributed to smaller structures, which can easily be described after some improvements in this procedure.

Figures 2 and 3GoGo are autoradiographic images from 8 different regions of a male and a female rat brain treated with 14C-CB 153 and Figure 4Go is the histologically stained (Luxol Fast Blueand Cresyl Violet) brain sections of the male rat which are shown in Figures 2 and 3GoGo. Localization of 14C-CB 153 and/or its metabolite(s) is reflected by black areas within each section of the brains, which were detected by phosphor imaging and corresponded to higher concentrations of the radioactive substance(s). The intraperitoneally injected 14C-CB 153 was distributed throughout the brain, both the gray and white tissues throughout the cerebral hemispheres, the brain stem, and the cerebellum. This was evident from the fact that all regions of each brain section were distinguishable from the background as compared to controls and 14C-CB 169-treated brain sections, which were indistinguishable from the background. However, each of the autoradiographic images showed that 14C-CB 153 was non-uniformly distributed within rat brains. Anatomical structures within each section accumulating higher concentrations of 14C-CB 153 were confirmed by histological staining of the sections following phosphor imaging and are presented in Figure 4Go to further the fact. Darker areas representing higher concentrations of 14C-CB 153 were found in corpus callosum, internal and external capsules, medial lemniscus, tegmentum of the mesencephalon, and metencephalon and cerebellar peduncles (Figs. 2–4GoGoGo). Most of the darker areas were associated with fiber tracts throughout the brain. The ventricular system and vascular spaces were devoid of any detectable 14C-CB 153-associated radioactivity (Figs. 2–4GoGoGo). Autoradiographic images of female rat brain sections shown in Figures 2 and 3GoGo are much lighter than the corresponding male sections. This finding was due to the fact that the females accumulated an average of 38% lower (difference between the two, shown in Figs. 2 and 3GoGo, was 28%) 14C-CB 153-associated brain radioactivity than males (Table 2Go). The method described in this article to determine localization of radiolabeled chemical within an organ was adequately sensitive to pickup this difference.



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FIG. 2. Phosphor images of 16 µm cryosections taken from different regions of fore- and mid-brain of a male and a female rat 48 h after a single intraperitoneal dose of 14C-CB 153 showing non-uniform distribution of radioactivity to different anatomic regions of the brain. Lyophilized cryosections were exposed to a phospho-imager storage screen for 75 days to achieve the images. Each section corresponds to the following approximate anterioposterior distance from bregma of adult male rats as shown in the Rat Brain Atlas (Paxinos and Watson, 1982Go): A and E, ~1.70 mm anterior; B and F, ~1.80 mm posterior; C and G, ~3.60 mm posterior; and D and H, ~3.80 mm posterior of bregma. Abbreviations: 3v, third ventricle; cc, corpus callosum; ec, external capsule; fmi, forceps minor corpus callosum; ic, internal capsule; LV, lateral ventricle; ml, medial lemniscus; rf, rhinal fissure.

 


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FIG. 3. Phosphor images of 16 µm cryosections taken from different regions of mid and hind brain of a male and a female rat 48 h after a single intraperitoneal dose of 14C-CB 153 showing non-uniform distribution of radioactivity to different anatomical regions of the brain. Lyophilized cryosections were exposed to a phospho-imager storage screen for 75 days to achieve the images. Each section corresponds to the following approximate anterioposterior distance from bregma of adult male rats as shown in the Rat Brain Atlas (Paxinos and Watson, 1982Go): A and E, ~4.52 mm posterior; B and F, ~6.72 mm posterior; C and G, ~10.80 mm posterior; and D and H, ~11.00 mm posterior of bregma. Abbreviations: fmj, forceps major corpus callosum; icp, inferior cerebellar peduncle; tm, tegmentum of the mesencephalon.

 


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FIG. 4. Histologically stained brain sections from the male rat shown in Figures 2 and 3GoGo for region identification. Selected structures are labeled for clarity of orientation to Figures 2 and 3GoGo. Abbreviations: 3v, 3rd ventricle; 4v, 4th ventricle; Aq, cerebral aqueduct; cc, corpus callosum; cg, cingulum; cn, cochlear nerve; cp, cerebral peduncle; CPu, caudate putamen; ec, external capsule; Ent, entorhinal cortex; fi, fimbria hippocampus; fmi, forceps minor corpus callosum; fmj, forceps major corpus callosum; hc, hippocampus commissure; ic, internal capsule; icp, inferior cerebellar peduncle; lc, lateral cerebellar nuclei; LV, lateral ventricle; mt, mammillothalamic tract; sp5, spinal trigeminal tract; tm, tegmentum of the mesencephalon.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, prepubertal rats were exposed intraperitoneally to 2 structurally distinct hexachlorobiphenyls, non-ortho coplanar CB 169 and di-ortho non-coplanar CB 153. These two HxCBs exert their toxicities by different mechanisms: the toxicity of CB 169 is mostly Ah receptor-mediated, whereas CB 153 acts through a variety of Ah receptor-independent mechanisms. Differences in tissue distribution further separate these congeners in terms of potencies and efficacies for similar responses.

The pattern of distribution of the two 14C-HxCBs and/or their metabolites within different regions of the brain was determined in serial sections by phosphor imaging which is at least 10 times more sensitive in detecting radioactivity than conventional X-ray film autoradiography. Around 9-fold higher concentrations of the non-coplanar CB 153 were found in brains than the coplanar CB 169 (Table 2Go), and this revealed an interesting non-uniform distribution of CB 153 to certain brain structures. On the other hand, levels of CB 169 in the brains were so low that no phosphor image could be achieved, even after ~5 times longer exposure (75 days for CB 153 versus 350 days for CB 169). These results are in agreement with previous findings that non-coplanar CBs are preferentially accumulated in rat brain with concentrations ranging in orders of magnitude higher than coplanar congeners (Kodavanti et al., 1998Go; Ness et al., 1994Go; Seegal et al., 1990Go, 1991Go; Sparling and Safe, 1980Go; Saghir et al., 1999Go). However, the specificity of congeners for different structures within the brain could not be demonstrated in any of the previous studies.

The present study indicates, for the first time, that 14C-CB 153 and/or its metabolite(s) accumulate, non-uniformly in the brains of male and female rats, with higher concentrations of CB153-derived radioactivity associated with specific structures within brains (Figs. 2 and 3GoGo). The specificity of 14C-CB 153 to different brain structures was confirmed by histological staining (Fig. 4Go). Higher radioactivity associated with CB 153 was found in fiber tracts of the telencephalon, brainstem, and cerebellum with no accumulation in the vascular spaces or the ventricles (Figs. 2–4GoGoGo).

Coplanar CBs do not accumulate appreciably in prepubertal rat brains (Saghir et al., 1999Go). In addition to their low brain accumulation, the brain-accumulated radioactivity of coplanar CB 77 (3,3',4,4'-tetraCB) was also found to be restricted to vascular spaces, whereas non-coplanar CB 47 (2,2',4,4'tetraCB) appeared to be homogeneously distributed throughout the brain tissues (Ness et al., 1994Go). CB 77 is rapidly metabolized and relatively high blood:fat ratios are reported for this congener because of the sequestration of metabolites in the blood (Abdel-Hamid et al., 1981Go; Klasson-Wehler et al., 1989Go; Mizutani et al., 1977Go; Saghir et al., 1999Go; Shimada and Sawabe, 1984Go). Thus, poorly metabolized CB 169 would be unlikely to accumulate even in the vasculature of the brain. Since coplanar CBs do not transfer from the vascular compartment into the brain and are inactive toward several neurochemical endpoints such as calcium buffering (Kodavanti et al., 1996Go), PKC translocation (Kodavanti et al., 1995Go), ryanodine receptor activation (Wong and Pessah, 1996Go), and biogenic amine dysregulation (Shain et al., 1991Go), these CB congeners may exert effects in the brain by indirect mechanisms, possibly through alteration in thyroid hormone homeostasis (Cheek et al., 1999Go; Hansen and Foley, 1997Go).

Both CB 153 and CB 169 significantly increased the liver weights in males and females, within 48 h following single ip doses of 8 mg/kg. The coplanar CB 169 increased liver weight to a greater extent (Fig. 1Go), consistent with known structure:activity relationships (Safe, 1994Go). In similar acute dosing protocols (2 doses, ip), non-coplanar CBs and mixtures with low TCDD equivalents (TEQ) were also less potent and efficacious than high TEQ mixtures in causing liver hypertrophy (Li and Hansen, 1996aGo; Li et al., 1998Go). Radioactivity associated with CBs 153 and 169 was sequestered similarly in fat, but coplanar CB 169 was decidedly more highly sequestered in liver (4–5% dose/g) than was the non-coplanar CB 153 (1–1.6% dose/g) (Table 2Go). A 3–4 fold higher liver accumulation of the parent CB 169 than CB 153 has been reported previously (Sparling and Safe, 1980Go). Using a protocol similar to this study, 20–50% of the intraperitoneal dose of coplanar CB 126 (3,3',4,4',5-pentaCB) was sequestered in the liver, while only 2–3% of non-coplanar CB 110 (2,3,3',4',6-pentaCB) was in the liver (Li et al., 1998Go). The more substantial increase of liver weight coupled with enhanced liver accumulation in rats treated with CB 169 (Fig. 1Go, Table 2Go) may have contributed to the reduced accumulation of this congener in the brains.

The most universally held concepts regarding PCB disposition in rats derive from the studies of Matthews and colleagues (Abdel-Hamid et al., 1980; Matthews and Tuey, 1980Go) who used young adult male Sprague-Dawley rats. It is clear that the weanling rats in this study differed from these models, but it is not clear whether the differences arise mainly from age or from strain. Weanling rats in this study were quite less efficient in excreting both of the HxCBs (Table 1Go) when compared with adult rats. In adult male Sprague-Dawley rats, 3.8% of the iv-dosed CB 153 was excreted during the initial (2–3 day, comparable to this study) excretion phase with a half-life of 1.17 days (Matthews and Tuey, 1980Go). At this rate, about 2.6% of the dose should have been excreted in the 2 days following the ip-dosing in the present study. However, the immature male rats excreted less than 25% of this amount and the immature females less than 12.5% (Table 1Go). Even for these slowly metabolized HxCBs, the lower excretion might be due to the fact that prepubertal rats do not have a drug metabolizing enzyme system as efficient as that of adult rats (Klinger et al., 1981Go; Lucier, 1978Go). The lack of efficient excretion of CBs by immature weanling rats makes them more vulnerable, and thus, even more appropriate for studying CB distribution to sensitive tissues.

Proportional tissue distribution, on the other hand, was not dramatically different from what would be expected in adult animals. There were modest sex differences. As predicted, CB 169 achieved 3-fold higher liver concentrations than CB 153. TCDD-like compounds, including coplanar CBs, are selectively retained in liver and this has been determined to be mainly accounted for by their binding to the highly induced CYP 1A2 (Diliberto et al., 1995Go; 1997). At TEQs similar to that presented by the CB 169 dose in this study, CYP 1A activities were induced dramatically in weanling female rats within the same short time frame as in the current study (Li and Hansen, 1996bGo). Thus, the CYP 1A2 binding could easily account for the higher liver residue of CB 169 than of CB 153.

The most important finding of this study is the non-uniform distribution of CB 153 and its association with specific structures within rat brains, which was described for the first time. This study also confirmed anticipated slower excretion of CBs by immature rats. Tissue accumulation of CBs in these rats was also more variable, probably due to the rapid phase of development and thus, more dynamic system.

One of the other important findings is the disproportionate retention of radioactivity in the mesenteric fat surrounding the injection site (Table 2Go), which indicates incomplete redistribution of the HxCBs within 2 days following intraperitoneal injection. This incomplete redistribution would have a profound effect on the short-term bioassays (2–3 days after exposure), which use the intraperitoneal route for animal exposure. This would lead to the underestimation of efficacies and potencies due to sequestration of large quantities of the administered doses into the peritoneal cavity, so that less than anticipated would reach the target tissues. For example, in the female rat integrated endocrine disruption assay (FRIEDA) (Li and Hansen, 1996bGo, 1997Go; Li et al., 1994Go), much of the intraperitoneal dose would still be sequestered in the peritoneal cavity when the animals are killed 20 h after the second dose. Although, significant effects have been observed on thyroid morphometry (Saeed and Hansen, 1997Go) as well as changes in serum thyroxin, enzyme induction, and uterotropic effects (Li and Hansen, 1996bGo; Li et al., 1994Go), responses might not have reached maximal levels due to sequestration of large quantities of the doses in mesenteric fat. Subcutaneous dose delivery probably results in delayed distribution from subcutaneous fat as well.

Even though experiments under steady-state concentrations of exposure are needed to accurately describe the net toxic effects of a chemical, it is important to determine the initial effects of pulsatic exposures in animals. This is especially important in developing animals where effects may not be apparent until later developmental stages, when the record of exposure no longer exists. This brief study is an initial step in describing the distribution of toxicants that may be relevant in defining events leading to developmental changes related to pulsatile exposures. The structure-specific brain accumulation of non-coplanar CBs (CB 153) demonstrated in this study should also be useful in further clarifying different responses to non-coplanar and coplanar CBs. Neurological effects are long-lasting and can even be permanent, if exposure occurs in animals that are still growing (e.g., neonates, prepubertal, etc.). Taking the transient sequestration of the ip dosed CBs in mesenteric fat that occurred in this study into consideration would also improve interpretation of short-term bioassays for lipophilic chemicals using the ip and subcutaneous routes of exposure.


    ACKNOWLEDGMENTS
 
This work was supported by U. S. Environmental Protection Agency, Contract 5D1420NAEX. The authors thank Drs. Janet Diliberto, Karl Jensen, Daniel Ness, and Hugh Tilson, for their comments on an earlier version of this manuscript. Authors also thank Don Warn, Patrick Moonasar, and Dr. Wohaib Hasan for their help with imaging and organizing figures.


    NOTES
 
The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

1 To whom correspondence should be addressed at Battelle, Pacific Northwest National Laboratories, P.O. Box 999, Mail Stop P7-56, Richland, WA 99352. Fax: (509) 376-7568. E-mail: shakil.saghir{at}pnl.gov. Back

This work was presented in part at the 36th Annual Meeting of the Society of Toxicology, Cincinnati, OH.


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