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Congener-Specific Distribution of Polychlorinated Biphenyls in Brain Regions, Blood, Liver, and Fat of Adult Rats Following Repeated Exposure to Aroclor 1254
TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.
153, 199 –210 (1998)
TO988534
Congener-Specific Distribution of Polychlorinated Biphenyls in Brain Regions, Blood, Liver, and Fat of Adult Rats Following Repeated Exposure to Aroclor 12541,2 Prasada Rao S. Kodavanti,* Thomas R. Ward,* Ethel C. Derr-Yellin,* William R. Mundy,* Ann C. Casey,‡ Brian Bush,†,‡ and Hugh A. Tilson* *Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, †Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201, and ‡School of Public Health, University at Albany, Albany, New York 12201– 0509 Received December 3, 1997; accepted July 17, 1998
Congener-Specific Distribution of Polychlorinated Biphenyls in Brain Regions, Blood, Liver, and Fat of Adult Rats Following Repeated Exposure to Aroclor 1254. Kodavanti, P. R. S., Ward, T. R., Derr-Yellin, E. C., Mundy, W. R., Casey, A. C., Bush, B., and Tilson, H. A. (1998). Toxicol. Appl. Pharmacol. 153, 199 –210. Our previous in vitro studies with both isolated organelles and primary neuronal cell cultures found that intracellular signal transduction can be perturbed by some noncoplanar PCBs at exposure levels of <10 mM. However, it is not clear whether such concentrations are achievable in brain in vivo. Also, the pattern of congener disposition and quantities of the PCB accumulation in tissues of animals exposed to commercial PCB mixtures is not well studied. In the present study, we have conducted PCB congenerspecific analysis in different brain regions, liver, blood, and fat of adult male Long–Evans rats dosed orally with Aroclor 1254 (0 or 30 mg/kg/day; once per day, 5 days/week for 4 weeks) in corn oil. Twenty-four hours after the last dose, rats were euthanized, and the brains were removed and dissected to obtain cerebellum, frontal cortex, and striatum. Liver, blood, and fat samples were also collected at the same time. Congener-specific analysis of PCBs was performed by high-resolution gas chromatography with electron capture detection. While PCB concentrations in control rat brain regions were less than 0.02 ppm, total PCB congeners in treated animals accumulated to much higher levels. Total levels in the frontal cortex, cerebellum, and striatum were 15.1 6 0.3, 13.1 6 1.7, and 8.2 6 2.6 ppm, respectively. The levels of PCBs in the fat, liver, and blood were 0.041, 0.002, and 0.001 ppm in control rats and 552, 38.3, and 1.6 ppm in treated rats, respectively. In addition to the differential total uptake between tissues, there was differential accumulation of PCBs with respect to the number of chlorines. In all the tissues, the more lightly chlorinated (tetra- and 1
Presented at the Annual Meeting of Society For Neuroscience, Washington, D.C., November 1996. (Soc. Neurosci. Abstr. 22, 1910, 1996). 2 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.
penta-) congeners accumulated less than their respective proportions in the parent Aroclor 1254 mixture. On the other hand, heavily chlorinated (hexa- to nona-) congeners accumulated more than the proportion of these congeners found in Aroclor 1254 mixture. This shift toward accumulation of heavily chlorinated congeners seems to be more pronounced in the brain than liver and fat. Predominant congeners (5–32% of total PCBs) detected in different brain regions, blood, liver, and fat are: 2,3,3*,4*,5,6- (no. 163) 1 2,2*,3,4,4*,5- (no. 138) (coeluted); 2,2’,4,4*,5,5*- (no. 153) 1 2,2*,3,3*,4,6*- (no. 132) (coeluted); 2,3,3*,4,4*,5- (no. 156) 1 2,2*,3,3*,4,4*,6- (no. 171) (coeluted); 2,3*,4,4*,5- (no. 118); 2,2*,4,4*,5-(no. 99); and 2,3,3*,4,4*- (no. 105). These congeners together accounted for about two thirds of the total PCB load in brain. All these predominant congeners are ortho-substituted and therefore are noncoplanar in nature. The total PCB concentrations accumulated in brain were as high as 50 mM (based on average molecular weight of 326.4 for Aroclor 1254) and, at these concentrations, intracellular second messengers were significantly affected in neuronal cultures and brain homogenate preparations in vitro. These results indicate that concentrations that altered Ca21 disposition and second messenger systems in vitro are achievable in brain in vivo following repeated exposure. © 1998 Academic Press
Polychlorinated biphenyls (PCBs) are persistent environmental contaminants and have become distributed throughout the entire ecosystem. Human populations have been exposed to these chemicals by both chronic and acute exposures. Chronic exposures have occurred as low level environmental exposures as well as high level occupational exposures (Kuwabara et al., 1978; Fischbein et al., 1979; Smith et al., 1982). Acute exposures have occurred through accidental ingestion (Masuda et al., 1982; Kuratsune and Shapiro, 1984). PCBs, which exist as 209 possible congeners, have been reported to cause a variety of effects including immunologic, teratogenic, reproductive, carcinogenic, and neurological effects (Kafafi et al., 1993; Seegal and Schantz, 1994). PCBs are not only a potential health threat to adults, but also to developing fetuses and
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0041-008X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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infants, because they cross the human placenta (Masuda et al., 1979) and are found in human milk (U.S. EPA, 1993; WHO, 1993). The effects elicited by PCBs seem to be dependent both on the structural characteristics of the congener (McKinney and Waller, 1994; Fischer et al., 1998) and on the time period of exposure (WHO, 1993). Current risk assessment for PCBs has been based on information from commercial PCB mixtures. Congener-specific actions have long been known (Hansen, 1987, Hansen, 1998; Safe, 1994) and workshops conducted by U.S. EPA and European Environmental Research Organization confirmed that congener-specific information is needed to better evaluate the risk potential of these compounds (U.S. EPA, 1993; Brouwer et al., 1995). Our laboratory explored the cellular mechanism(s) for the neurological effects of PCBs in primary neuronal cell cultures as well as brain homogenates. Our in vitro studies indicated that calcium homeostasis and other second messenger systems including inositol phosphate and protein kinase C (PKC) translocation are sensitive to noncoplanar PCBs at concentrations of #30 mM where cytotoxicity as measured by LDH leakage was not detected (Kodavanti and Tilson, 1997). These studies have been extended to in vivo in adult rats following repeated exposure to a commercial PCB mixture. The changes in second messenger systems seen in vivo (Kodavanti et al., 1998) are similar to those reported in vitro in neuronal preparations. In this study, we conducted congener-specific analysis of PCBs in order to determine (1) if concentrations of PCBs that altered second messenger systems in vitro are achievable in vivo, (2) if the profile of congeners in brain and other tissues reflect the composition of the parent mixture, and (3) if there is a differential PCB accumulation in different brain regions. MATERIALS AND METHODS Chemicals. A commercial PCB mixture, Aroclor 1254, (purity . 99%; lot no. 6024) was purchased from AccuStandard Inc. (New Haven, CT) for dosing rats. Aroclors 1221, 1016, 1254, and 1260 standards were obtained from Ultra Scientific (North Kingstown, RI). Florisil was obtained from U.S. Silica (Pittsburgh, PA). The solvents used in extraction of samples were nanograde from Mallinckrodt, Inc. (St. Louis, MO). All other chemicals used in the assays were obtained from commercial sources. Animals. Adult male (60 days; 250 –270 g) Long–Evans hooded rats were obtained from Charles River Laboratory (Raleigh, NC) and housed two per cage in American Association for Accreditation of Laboratory Animal Care (AAALAC) approved animal facilities. All experiments were approved in advance by the National Health and Environmental Effects Research Laboratory animal care committee of the U.S. EPA. Food and water were provided ad libitum. Temperature was maintained at 21 6 2°C and relative humidity was maintained at 50 6 10% with a 12-h light/dark cycle (light 7:00 to 19:00 h). In vivo dosing. After 1 week of acclimation, rats were dosed by oral gavage with Aroclor 1254 in corn oil (2 ml/kg). The selected dosages were 0 or 30 mg/kg/day. The rats (four rats per group) were dosed five times per week for 4 weeks. The dosing time was always between 8:00 and 10:00 a.m. The animals were weighed three times per week. Twenty-four hours after the last dosage, rats were euthanized and blood was collected. Liver slices (about 1 g) were collected from the main lobes and fat was collected from the abdomen. Different brain regions (cerebellum, frontal cortex, and striatum) were dis-
sected as described previosly (see companion paper, Kodavanti et al., 1998). All the samples were frozen on dry ice, and stored at 280°C until congenerspecific analysis of PCBs. Neurochemical measurements were made in the same batch of animals (see companion paper, Kodavanti et al., 1998) Extraction of PCB congeners in tissues and blood. Congener-specific analysis of PCBs was performed using a high-resolution gas chromatography with electron capture detection (Bush et al., 1985, 1989). Briefly, tissue samples were mixed with anhydrous sodium sulfate to remove water. The tissue sample and sodium sulfate were then ground together with 20 ml of hexane:acetone (1:1) in a SDT tissumizer (Tekmar, Cincinnati, OH) for 1 min. The supernatant hexane was decanted into a Kuderna-Danish evaporator. The extraction was repeated twice more and the combined extracts were concentrated to approximately 2 ml on a steam bath. For extraction of PCBs from whole blood, an equal amount of methanol was added and the tube contents were shaken vigorously. The mixture was left for 15 min in order to break down the proteins and eliminate any blood borne pathogens. To this mixture, 15 ml of diethylether:hexane (1:1) was added and the tubes were shaken again. When the layers separated, the upper layer was pipetted into a Kuderna-Danish evaporator. The extraction was repeated two more times and the combined extracts were concentrated to approximately 1 ml. A 0.1-ml aliquot was used for lipid analysis and the rest of the sample was processed as per tissues.
TABLE 1 Congener-Specific Analysis of PCBs in Water and Rat Chow Used in Aroclor 1254 Study
Water/chow Water
Rat chow
PCB congener Total PCBs 2, 49 and 2, 3 2, 29, 4, 49 and 2, 29, 4, 5 Total PCBs 2, 4 and 2, 5 2, 49 and 2, 3 2, 29, 5, 59 2, 29, 3, 59 2, 29, 3, 5, 69 and 2, 4, 49, 5 2, 39, 49, 5 2, 39, 4, 49 2, 29, 4, 5, 59 2, 29, 4, 49, 5 2, 29, 39, 4, 5 2, 29, 3, 4, 59 3, 39, 4, 49 and 2, 3, 39, 49, 6 2, 29, 3, 39, 5, 69 2, 29, 3, 49, 59, 6 2, 39, 4, 49, 5 2, 29, 3, 49, 5, 59 2, 29, 4, 49, 5, 59 and 2, 29, 3, 39, 4, 69 2, 3, 39, 4, 49 2, 29, 3, 4, 49, 59 2, 29, 3, 39, 4, 5, 6, 69 DDE Mirex
IUPAC no.
8 5 47 48 7 9 8 5 52 44 94 74 70 66 101 99 97 87 77 110 135 149 118 146 153 132 105 138 200
Concentration (ppb; ng/g or ml) 1.422 0.986 0.436 4.730 0.457 0.227 0.258 0.137 0.140 0.317 0.294 0.420 0.315 0.108 0.076 0.380 0.155 0.124 0.221 0.042 0.205 0.080 0.479 0.295 0.253 0.757
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TABLE 2 Different PCB Congeners Based on the Number of Chlorines Detected in Different Brain Regions, Blood, Liver, and Fat of Adult Rats Dosed with Aroclor 1254 PCB congeners (ppb; ng/g tissue or ml blood) Tissue
Mono, di- and tri-
Tetra-
Penta-
Hexa-
Hepta-
Octa- and nona
Aroclor 1254 Brain Frontal cortex Cerebellum Striatum Blood Liver Fat
(2.1)
(19.1)
(49.6)
(25.9)
(2.9)
(0.5)
6 (,1) 2 (,1) 3 (,1) 0 (0) 0 (0) 0 (0)
542 (4) 577 (4) 223 (3) 47 (3) 180 (,1) 30321 (6)
3964 (26) 3652 (28) 2346 (28) 540 (35) 12850 (34) 249760 (45)
7970 (53) 7044 (54) 4464 (54) 746 (48) 21961 (57) 223275 (41)
2304 (15) 1584 (12) 1030 (13) 153 (10) 2116 (6) 33779 (6)
330 (2) 259 (2) 173 (2) 61 (4) 1166 (3) 14416 (2)
Note. Mean values are reported (n 5 3). The numbers in parenthesis indicate the percentage of total PCBs in that tissue. Dosage of Aroclor 1254 was 30 mg/kg/day; 5 days/week for 4 weeks.
PCB congener-specific analysis. The concentrated solution was then transferred quantitatively to a 1 3 15 cm chromatography column containing 10 g of calibrated 4% deactivated Florisil with a 2-g layer of sodium sulfate on top of the Florisil. The column was eluted with 50 ml of hexane containing PCBs, concentrated to 1 ml using a Kuderna-Danish evaporator, and pipetted to a 1 ml Wheaton GC vial for analysis. The samples were analyzed on a Hewlett-Packard 5890 gas chromatograph equipped with a Ni-63 electron capture detector with a fused silica column with cross-linked 5% phenylmethylsilicone coating (0.33 mm film thickness, 0.25 mm id). The oven temperature was held at 100°C for 2 min, and then raised up to 160°C at 10°C/min, then 1°C/min to 190°C, and then 2°C/min to a final temperature of 270°C. The temperature was held at 270°C for 10 min until all congeners were eluted. Approximately 113 PCB congeners were identified and verified using a separate Apiezon L capillary column (Bush et al., 1985). The microprocessor of the gas chromatograph was calibrated with a solution of Aroclors 1221, 1016, 1254, and 1260 (200 ng/ml of each) fortified with hexachlorobenzene (HCB), 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), and mirex at 5, 10, and 10 ng/ml, respectively, and 3,39,4,49-tetrachlorobiphenyl at 20 ng/ml. This mixture has been previously fully characterized (Bush et al., 1985; Schultz et al., 1989). Data analysis was carried out with an IBM compatible personal computer using FILESERVER and entered into LOTUS 123 (Lotus Development Corp., Cambridge, MA). Quality control for the analysis was ensured by running replicates of a sample in every batch of 10 samples that were analyzed to ensure the consistency of sample extraction. Replicate samples of the first set of fat, liver, and brain of each group were extracted and analyzed on a GC with a Hewlett Packard Ultra II and an Apiezon L column. The Ultra II is used as a primary column for quantitation and Apiezon L is used to confirm peak identities. The samples were spiked with DDE to standardize retention times and check percent recovery of the extraction process. The average minimum detectable level (MDL) (p , 0.05 that the reported level is zero) (Type 1 error) for a sample weighing 1 g was 0.02 ng/g (U.S. EPA, 1984). It should be noted that even at one quarter of this value, the chances for the presence of that congener is 87% (Type II error). The results of PCB analysis were expressed as ng or or mg PCB/g wet weight tissue or ml blood.
RESULTS
respectively (Table 1). The trace amounts of PCBs in rat chow and water are typical of the background levels reported previously (Shain et al., 1986) and will not interfere with the interpretation of results from the present experiment. The accumulation of PCBs in the control rat tissues was very low compared to the treated animals. The total PCB accumulation in the brains of control animals was less than 0.02 ppm. Other tissues also had relatively low levels, with the fat containing the highest level (0.041 ppm). In all control tissues, total PCBs and individual congeners were less than 1% of treated values. Different PCB Congeners in the Commercial Mixture, Aroclor 1254 (Lot No. 6024) The distribution of congeners in the Aroclor 1254 is shown in Tables 2, 4, and 5. In agreement with published reports TABLE 3 Toxicokinetic Distribution of PCB Congeners in Adult Rats Dosed with Aroclor 1254 Total PCBs (ppm; mg/g tissue or ml blood) Control
Brain Frontal cortex Cerebellum Striatum Blood Liver Fat
0.019a 0.004 0.000 0.001 0.002 0.041
Aroclor 1254
15.12 6 0.31b 13.13 6 1.68 8.24 6 2.57 1.55 6 0.01 38.27 6 8.22 551.55 6 41.06
Note. Dosage of Aroclor 1254 was 30 mg/kg/day; 5 days/week for 4 weeks. Since controls give information about background levels of exposure, which is minimal, only one animal was analyzed. b For Aroclor 1254-treated animals, three rats were analyzed and the values reported are means 6 SE of three rats. a
The rat chow and water given to control and exposed rats contained low levels of a number of PCB congeners (Table 1). Total PCBs in water and rat chow were 1.4 and 4.7 ppb,
Tissue
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TABLE 4 Congener-Specific Analysis of PCBs in Different Regions of Brain of Rats Treated with Aroclor 1254 Congener Total PCB congeners 3, 39, 4, 491 2, 3, 39, 49, 6 2, 29, 4, 5, 59 2, 39, 4, 49, 5 2, 29, 3, 39, 49, 5, 61 2, 29, 3, 4, 49, 59 2, 39, 4, 49 2, 29, 3, 59, 6 2, 39, 49, 5 2, 29, 4, 49, 5, 591 2, 29, 3, 39, 4, 69 2, 3, 39, 4, 49 2, 29, 4, 49, 5 2, 29, 39, 4, 5 2, 29, 5, 59 2, 29, 3, 49, 59, 6 2, 29, 3, 4, 59 DDE 2, 3, 4, 491 29, 3, 39, 4 2, 29, 3, 39, 4, 491 2, 39, 4, 49, 5, 59 2, 29, 3, 59 2, 29, 3, 5, 691 2, 4, 49, 5 2, 29, 3, 39, 4, 59 2, 3, 39, 4, 49, 6 2, 29, 3, 39, 4, 49, 61 2, 3, 39, 4, 49, 5 2, 29, 3, 39, 4, 5 2, 29, 3, 49, 5, 6 2, 29, 3, 39, 5, 69 2, 29, 4, 59 2, 29, 3, 4, 5, 591 2, 29, 3, 39, 5, 6, 69 2, 29, 3, 4, 49, 5, 59 2, 29, 3, 39, 4, 49, 51 2, 3, 39, 4, 49, 5, 6 2, 29, 3, 49, 5, 59 2, 3, 49, 6 2, 29, 3, 39, 4, 6, 69 2 2, 29, 3, 39, 6, 69 2, 29, 3, 5, 59, 61 2, 29, 3, 39, 4 2, 29, 3, 39, 5, 6 2, 49, 5 2, 29, 3, 39, 4, 59, 6, 69 2, 3, 39, 61 2, 29, 3, 491 3, 4, 49
IUPAC no.
771 110 101 118 1631 138 661 95 70 1531 132 105 99 97 52 149 87 601 56 1281 167 44 941 74 130 158 1711 156 129 147 135 49 1411 179 180 1701 190 146 64 176 1 136 1511 82 134 31 201 591 421 37
Aroclor 1254
Cerebellum
Frontal cortex
Striatum
100.00 13.21
100.00 0.07
100.00 0.07
100.00 0.02
9.94 9.81 7.87
0.43 12.30 19.21
0.40 11.63 18.43
0.40 12.94 19.42
5.53
1.69
1.17
1.96
5.11 4.52
1.70 17.24
1.59 16.57
0.00 17.10
4.38 4.32 3.42 2.86 2.80 2.57 2.34 2.27
4.93 8.93 0.10 0.00 0.41 0.49 0.68 0.49
4.79 8.31 0.09 0.00 0.27 0.51 0.64 0.47
4.81 9.15 0.10 0.00 0.20 0.41 0.65 0.46
2.10
0.73
0.70
0.76
1.46 1.26
0.00 0.99
0.00 0.66
0.00 0.88
1.22 1.16 1.16
0.94 0.32 10.66
0.93 0.47 11.12
0.93 1.16 10.38
1.02 0.86 0.86 0.74 0.69
0.30 0.03 0.00 0.00 0.02
0.38 0.02 0.01 0.00 0.02
0.30 0.14 0.12 0.00 0.05
0.68 0.65
1.98 3.39
2.39 3.43
2.89 3.19
0.65 0.60 0.56 0.54 0.50 0.49
2.42 0.00 3.31 0.00 0.00 0.01
2.40 0.00 6.20 0.00 0.00 0.04
2.33 0.00 3.16 0.00 0.00 0.00
0.49 0.47 0.38 0.30
2.08 0.00 1.53 0.00
2.12 0.00 1.57 0.00
2.00 0.00 1.68 0.00
Note. Dosage of Aroclor 1254 was 30 mg/kg/day; 5 days/week for 4 weeks. All values are % of total: total PCBs in cerebellum 5 13, 128 ng/g; frontal cortex 5 15, 118 ng/g; and striatum 5 8239 ng/g; n 5 3.
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TABLE 4—Continued Congener 2, 29, 3, 39, 4, 5, 69 2, 29, 3, 39 2, 29, 4, 491 2, 29, 4, 5 4 2, 29, 3, 49, 5, 59, 61 2, 29, 3, 4, 49, 5, 69 2, 29, 3, 4, 49, 59, 6 2, 29, 5 2, 49,1 2, 3 2, 29, 3, 39, 49, 5, 6 2, 29, 31 2, 49, 6 29, 3, 4 2, 291 2, 6 4, 491 2, 29, 4 2, 29, 3, 6 2, 3, 49 2, 29, 3, 39, 4, 5, 59 2, 39 2, 29, 3, 4, 5, 59, 6 2, 29, 3, 39, 4, 5, 69 2, 29, 3, 4, 49, 5, 59, 61 2, 29, 3, 39, 4, 49, 5, 69 2, 29, 3, 39, 4, 49, 5, 59 2, 29, 3, 39, 4, 5, 59, 69 2, 39, 5 2, 41 2, 5 HCB 2, 3, 61 2, 39, 6 2, 29, 6 2, 39, 4 2, 4, 49 Mirex 2, 29, 3, 39, 4, 49, 5, 6 2, 29, 3, 39, 4, 49, 5, 59, 6
IUPAC no. 174 40 471 48 3 1871 182 183 18 81 5 177 161 32 33 41 10 151 17 45 22 172 6 185 200 2031 196 194 199 26 71 9 241 27 19 25 28 195 206
Aroclor 1254
Cerebellum
Frontal cortex
Striatum
0.28 0.28 0.24
0.06 0.00 0.09
0.06 0.00 0.08
0.00 0.00 0.06
0.23 0.19
0.00 0.71
0.00 0.71
0.00 0.73
0.19 0.18 0.16
0.56 0.00 0.00
0.56 0.00 0.00
0.52 0.00 0.00
0.13 0.10
0.51 0.00
0.50 0.00
0.54 0.00
0.09 0.07
0.00 0.00
0.00 0.00
0.00 0.00
0.07
0.00
0.00
0.00
0.06 0.05 0.05 0.03 0.03 0.03 0.02
0.00 0.00 0.31 0.00 0.54 0.03 0.16
0.00 0.00 0.31 0.00 0.35 0.06 0.15
0.00 0.00 0.25 0.00 0.54 0.07 0.11
0.02 0.02 0.01 0.01
0.10 0.10 0.00 0.00
0.05 0.18 0.00 0.02
0.09 0.15 0.00 0.03
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.01 0.00 0.05 0.08
0.00 0.00 0.01 0.00 0.09 0.08
0.00 0.00 0.00 0.00 0.00 0.00
(Bush et al., 1985; Erickson, 1986; Schultz et al., 1989), Aroclor 1254 contained more ortho-substituted (noncoplanar) PCB congeners than non-ortho-substituted (coplanar) congeners. By our procedure, di-to-tetra-ortho-substituted PCBs (nondioxin like) accounted for 54.5% while mono-ortho PCBs (mixed type) were 45% of total PCBs in the Aroclor 1254 mixture. The only possible non-ortho (dioxin-like) congener detected was 3,39,4,49-tetrachlorobiphenyl (no. 77), which coeluted with 2,3,39,49,6-pentachlorobiphenyl (no. 110) and these two congeners together accounted for 13.2% of the total PCBs. Frame et al. (1996) reported that the coplanar PCB congeners (nos. 77, 126, and 169) were less than 1% of total PCBs in two different Aroclor 1254 mixtures. In this study, a special analysis with carbon separation of coplanar PCBs was not con-
ducted on these samples for reasons of cost. However, a separate analysis of this batch of Aroclor 1254 (lot no. 6024) by the Department of Chemistry at University of Kiel, Germany indicated that coplanar non-ortho substituted PCBs (nos. 77, 126, and 169) were 3% of total PCBs (Kodavanti et al., unpublished data). The variations in the composition of these congeners in Aroclor 1254 might be due to differences in the manufacturing process of these mixtures. PCB congenerspecific analysis also revealed that pentachlorobiphenyls were the highest (49.6% of total) followed by hexachlorobiphenyls (25.9% of total) and tetrachlorobiphenyls (19.1% of total). All other PCB congeners were less than 6% of total (Table 2). Of the 88 congeners or pairs (some coeluted), 12 congeners with IUPAC numbers of 77 1 110 (coeluted), 101, 118, 138 1 163
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KODAVANTI ET AL.
(coeluted), 66, 70, 153 1 132 (coeluted), 105, and 99 accounted for over 60% of the total mixture and this is in agreement with a recent report by Frame et al. (1996). DDE was the only major contaminant detected, which was 2.34% of the total mixture. Distribution of PCB Congeners in Different Brain Regions While PCB concentration in control rat brain regions was less than 20 ppb, total PCB congeners in exposed animals accumulated to ppm levels (Table 3). Total PCB congeners (means 6 SE) were greater in frontal cortex (15.1 6 0.3 ppm) and cerebellum (13.1 6 1.7 ppm) compared to striatum (8.24 6 2.57 ppm), suggesting marginal differences in the accumulation of PCBs in brain regions. Distribution of PCBs by number of chlorines in the Aroclor 1254 mixture and the various tissues is shown in Table 2. As expected, the percentage of the various congeners in Aroclor 1254 exhibited a bell-shaped curve with the peak at pentachlorobiphenyls (49.6% total). The distribution of PCBs by number of chlorines in brain is also bell shaped, but the peak was at hexachlorobiphenyls. There was a shift toward accumulation of more heavily chlorinated congeners in all three brain regions. The congeners with five or more chlorines accounted for 78.9% of the total in the Aroclor 1254 mixture while they were approximately 97% in the brain. The proportion of pentachlorobiphenyls was 49.6% in Aroclor 1254 while the accumulation of pentachlorobiphenyls was much lower (26 –28% of total) in brain regions. Although the total level of PCB congeners was slightly lower in the striatum, the ratios of congeners (by weight) among brain regions remained constant (Table 2). Predominant congeners detected in different brain regions were: 2,3,39,49,5,6- (no. 163) 1 2,29,3,4,49,5- (no. 138) (coeluted); 2,29,4,49,5,59- (no. 153) 1 2,29,3,39,4,69- (no. 132) (coeluted); 2,3,39,4,49,5- (no. 156) 1 2,29,3,39,4,49,6- (no. 171) (coeluted); 2,39,4,49,5- (no. 118), 2,3,39,4,49- (no. 105), and 2,29,4,49,5-(no. 99) (Table 4; Fig. 1). Total PCB concentrations observed in brain were as high as 15 ppm, equivalent to 40 to 50 mM based on average molecular weight of 326.4 for Aroclor 1254 mixture (Erickson, 1986) and, at these concentrations, intracellular second messengers were significantly affected in neuronal preparations in vitro (Kodavanti et al., 1993, 1994, 1995). Distribution of PCB Congeners in Blood, Liver, and Fat The levels of total PCBs in the fat were high (552 6 41 ppm). The circulating levels of total PCBs in the blood were 1.55 6 0.01 ppm. Liver accumulated greater amounts of PCBs (38.3 6 8.2 ppm) than any of the brain regions (Table 3). The shift toward accumulation of more heavily chlorinated congeners, as seen in the brain, was also observed in blood, liver, and fat, but to a lesser extent. Blood and liver contained 97 and 99% of the heavier ($penta-) congeners, respectively, compared to 78.9% in the Aroclor 1254 (Table 2). Accumulation of
heavier congeners in fat was slightly less than other tissues accounting for 94% of the total. Predominant congeners detected in blood, liver, and fat were PCBs with IUPAC number of 138 1 163 (coeluted), 118, 99, 105, and 132 1 153 (coeluted) (Table 5; Fig. 2). In addition, PCBs with IUPAC numbers of 156 1 171 were also detected in higher amounts (10% of total) in liver (Table 5; Fig. 2). DISCUSSION
We have previously demonstrated that second messenger systems including inositol phosphate, Ca21 homeostasis, and PKC translocation are preferentially sensitive to noncoplanar PCBs at low micromolar concentrations in neuronal cultures and brain homogenate preparations (Kodavanti and Tilson, 1997; Tilson and Kodavanti, 1997). Higher concentrations ($100 mM) and longer exposure periods (.1 h) were required to see any cytotoxicity. It is known that perturbations in Ca21 homeostasis and sustained activation of PKC are involved in the neurological effects of a variety of chemicals (Farber, 1990; Mattson, 1991). Consistent with our in vitro observations, the data from our in vivo study (Kodavanti et al., 1998) indicate that intracellular Ca21 buffering and PKC activity can be affected in brain regions of rats exposed repeatedly with a commercial PCB mixture, Aroclor 1254. However, it was not known whether concentrations required to see a significant change in vitro are achievable in vivo or the accumulation of congeners reflects the parent mixture or if there is a differential accumulation in brain regions. The present study demonstrated that, following repeated administration of Aroclor 1254, the brain can accumulate PCBs up to 15 ppm, which is equivalent to 40 to 50 mM based on the average molecular weight of 326.4 for Aroclor 1254 mixture (Erickson, 1986). These are the concentrations that have significant effects on second messenger systems in vitro using cell cultures or brain homogenate preparations (Kodavanti et al., 1993, 1994, 1995, 1996; Shafer et al., 1996). Therefore, PCB concentrations which alter calcium buffering and PKC activity in vitro are achievable in vivo. It is noteworthy that most of the congeners accumulated in brain are orthosubstituted and noncoplanar in nature. Similar observations were also made by Seegal and Shain (1992) where several mono- and di-ortho-substituted congeners were detected in rat brain following Aroclor 1254 exposure in diet. Interestingly, Dewailly et al. (1995) reported PCB levels in various tissues from human autopsies in Greenland and mean concentration of total PCBs were 16.9 mg/kg (ppm) in adipose tissue, 12.2 mg/kg in liver, and 2.0 mg/kg in brain lipids. The PCB congeners found at the highest levels in the Aroclor mixture, nos. 138 1 163 (coeluting), 118, 105, and 132 1 153 (coeluting) were also highest in the brain tissue, together accounting for almost 50% of the total. Other congeners nos. 77 1 110, 101, 66 1 95, and 70, which represent 33.8% of the Aroclor 1254 mixture, were less than 2% of the congeners
CONGENER-SPECIFIC DISTRIBUTION OF PCBS IN BRAIN
205
FIG. 1. Congener-specific accumulation of PCBs in different brain regions (frontal cortex, cerebellum, and striatum) of rats treated with Aroclor 1254 (30 mg/kg/day; 5 days/week for 4 weeks). The x-axis shows PCB concentrations in ng/g wet weight tissue (ppb). The y-axis shows IUPAC numbers of PCB congeners in the order of elution from the column. Only mean values (n 5 3) were plotted for clarity.
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TABLE 5 Congener-Specific Analysis of Polychlorinated Biphenyls in Blood, Liver, and Fat of Rats Treated with Aroclor 1254 Congener Total PCB congeners 3, 39, 4, 49,1 2, 3, 39, 49, 6 2, 29, 4, 5, 59 2, 39, 4, 49, 5 2, 3, 39, 49, 5, 61 2, 29, 3, 4, 49, 59 2, 39, 4, 49 2, 29, 3, 59, 6 2, 39, 49, 5 2, 29, 4, 49, 5, 59, 1 2, 29, 3, 39, 4, 69 2, 3, 39, 4, 49 2, 29, 4, 49, 5 2, 29, 39, 4, 5 2, 29, 5, 59 2, 29, 3, 49, 59, 6 2, 29, 3, 4, 59 DDE 2, 3, 4, 491 29, 3, 39, 4 2, 29, 3, 39, 4, 49,1 2, 39, 4, 49, 5, 59 2, 29, 3, 59 2, 29, 3, 5, 691 2, 4, 49, 5 2, 29, 3, 39, 4, 59 2, 3, 39, 4, 49, 6 2, 29, 3, 39, 4, 49, 61 2, 3, 39, 4, 49, 5 2, 29, 3, 39, 4, 5 2, 29, 3, 49, 5, 6 2, 29, 3, 39, 5, 69 2, 29, 4, 59 2, 29, 3, 4, 5, 591 2, 29, 3, 39, 5, 6, 69 2, 29, 3, 4, 49, 5, 59 2, 29, 3, 39, 4, 49, 51 2, 3, 39, 4, 49, 5, 6 2, 29, 3, 49, 5, 59 2, 3, 49, 6 2, 29, 3, 39, 4, 6, 69 2 2, 29, 3, 39, 6, 69 2, 29, 3, 5, 59, 61 2, 29, 3, 39, 4 2, 29, 3, 39, 5, 6 2, 49, 5 2, 29, 3, 39, 4, 59, 6, 69 2, 3, 39, 61 2, 29, 3, 49 3, 4, 49
IUPAC no.
771 110 101 118 1631 138 661 95 70 1531 132 105 99 97 52 149 87 601 56 1281 167 44 941 74 130 158 1711 156 129 147 135 49 1411 179 180 1701 190 146 64 176 1 136 1511 82 134 31 201 591 421 37
Aroclor 1254
Blood
Liver
Fat
100.00 13.21
100.00 0.49
100.00 0.00
100.00 1.57
9.94 9.81 7.87
0.74 13.63 22.62
0.00 13.03 32.12
2.22 17.80 18.24
5.53
1.53
0.00
3.17
5.11 4.52
0.07 18.59
0.00 18.00
0.32 12.41
4.38 4.32 3.42 2.86 2.80 2.57 2.34 2.27
8.32 10.80 0.20 0.00 0.00 0.20 0.24 0.59
8.04 14.37 0.00 0.00 0.00 0.00 0.25 0.00
7.58 13.65 0.61 0.00 1.14 0.96 1.77 1.11
2.10
1.02
0.00
2.19
1.46 1.26
0.00 1.33
0.00 0.49
0.00 1.84
1.22 1.16 1.16
0.91 2.19 0.61
0.00 0.00 10.05
1.04 3.14 0.41
1.02 0.86 0.86 0.74 0.69
0.00 0.07 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.07 0.11 0.00 0.00 0.00
0.68 0.65
3.80 3.49
2.72 2.47
2.01 1.96
0.65 0.60 0.56 0.54 0.50 0.49
2.51 0.00 0.00 0.00 0.00 0.00
1.67 0.00 0.00 0.00 0.00 0.00
2.21 0.00 0.00 0.00 0.00 0.00
0.49 0.47 0.38 0.30
0.00 0.00 3.31 0.00
0.00 0.00 3.24 0.00
0.00 0.00 2.65 0.00
Note. Dosage of Aroclor 1254 was 30 mg/kg/day; 5 days/week for 4 weeks. All values are % of total: total PCBs in blood 5 1547 ng/g; liver 5 38,273 ng/g; fat 5 551,551 ng/g; n 5 3.
207
CONGENER-SPECIFIC DISTRIBUTION OF PCBS IN BRAIN
TABLE 5—Continued Congener 2, 29, 3, 39, 4, 5, 69 2, 29, 3, 39 2, 29, 4, 491 2, 29, 4, 5 4 2, 29, 3, 49, 5, 59, 61 2, 29, 3, 4, 49, 5, 69 2, 29, 3, 4, 49, 59, 6 2, 29, 5 2, 49,1 2, 3 2, 29, 3, 39, 49, 5, 6 2, 29, 31 2, 49, 6 29, 3, 4 2, 291 2, 6 4, 491 2, 29, 4 2, 29, 3, 6 2, 3, 49 2, 29, 3, 39, 4, 5, 59 2, 39 2, 29, 3, 4, 5, 59, 6 2, 29, 3, 39, 4, 5, 6, 69 2, 29, 3, 4, 49, 5, 59, 61 2, 29, 3, 39, 4, 49, 5, 69 2, 29, 3, 39, 4, 49, 5, 59 2, 29, 3, 39, 4, 5, 59, 69 2, 39, 5 2, 41 2, 5 HCB 2, 3, 61 2, 39, 6 2, 29, 6 2, 39, 4 2, 4, 49 Mirex 2, 29, 3, 39, 4, 49, 5, 6 2, 29, 3, 39, 4, 49, 5, 59, 6
IUPAC no.
Aroclor 1254
Blood
Liver
Fat
0.28 0.28 0.24
0.00 0.00 0.09
0.00 0.00 0.00
0.00 0.00 0.15
0.23 0.19
0.00 0.83
0.00 0.00
0.00 0.47
0.19 0.18 0.16
0.58 0.00 0.00
0.00 0.00 0.00
0.34 0.00 0.00
0.13 0.10
0.55 0.00
0.00 0.00
0.36 0.00
0.09 0.07
0.00 0.00
0.00 0.00
0.00 0.00
0.07
0.00
0.00
0.00
0.06 0.05 0.05 0.03 0.03 0.03 0.02
0.00 0.00 0.00 0.00 0.32 0.02 0.06
0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.30 0.00 0.00
0.02 0.02 0.01 0.01
0.24 0.19 0.00 0.00
0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.10
0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00
174 40 471 48 3 1871 182 183 18 81 5 177 161 32 33 41 10 151 17 45 22 172 6 185 200 2031 196 194 199 26 71 9 241 27 19 25 28 195 206
accumulated in the brain, suggesting that these congeners do not preferentially accumulate in brain, probably due to liver metabolism (Dragnev et al., 1994). On the other hand, no. 99, which was 4.3% of the Aroclor 1254, accounted for about 8 to 9% of the total brain congeners. Similarly, no. 156 1 171, which was only 1.2% of the Aroclor 1254, accounted for about 10 to 11% of the total brain congeners, suggesting preferential accumulation due to less metabolism. In the Greenland study, major congeners detected in human brain were PCBs 99, 105, 118, 138, 153, and 156, which are similar to the ones reported in the present study. PCBs 118, 138, and 153 have been detected in serum samples from U.S. population and Yusho patients (Patterson et al., 1994; Schecter et al., 1994). These same PCB congeners were present at the highest concentra-
tions in frontal cortex, cerebellum, and striatum of Aroclor 1254-dosed rats. Several PCB congeners (IUPAC nos. 99, 118, 138, 153, and 156) that significantly affected intracellular calcium buffering (Kodavanti et al., 1996) and PKC translocation in vitro in neuronal cultures (Kodavanti et al., 1995) are present in the parent Aroclor 1254 mixture. These congeners also accumulated at higher concentrations in brain regions, suggesting a significant role for these congeners in the neurotoxicity of PCBs. Although the congener distribution is similar between the brain regions, the frontal cortex and cerebellum accumulated PCBs to a slightly higher level than did the striatum. The PCB levels for the frontal cortex, cerebellum, and striatum are 15.1,
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FIG. 2. Congener-specific accumulation of PCBs in blood, liver, and fat tissues of rats treated with Aroclor 1254 (30 mg/kg/day; 5 days/week for 4 weeks). The x-axis shows PCB concentrations in ng/g wet weight tissue or ml blood (ppb). The y-axis shows IUPAC numbers of PCB congeners in the order of elution from the column. Only mean values (n 5 3) were plotted for clarity.
CONGENER-SPECIFIC DISTRIBUTION OF PCBS IN BRAIN
13.1, and 8.2 mg/g wet weight tissue, respectively. Such minor differences in accumulation of PCBs in different brain regions was also reported previously (Seegal et al., 1991). However, Ness et al. (1994) reported no differences in PCB concentrations among brain regions, but the different congeners differed from each other in degree of bioaccumulation in rats developmentally exposed to another Aroclor mixture (Aroclor 1242). The minor differences in the accumulation of PCBs in brain regions, as seen in the present study, cannot be explained simply by differences in lipid content. Gospe and Calaban (1988) reported that total lipid content (as % of wet weight) is similar in all three brain regions (8.9, 8.6, and 8.7 for the frontal cortex, cerebellum, and caudate, respectively). The PCB congener-specific distribution in blood, liver, and fat was qualitatively similar to that of brain regions (Tables 4 and 5). Most of the congeners that accumulated are orthosubstituted and noncoplanar in nature. Also, this study agrees with the typical behavior of highly lipophilic compounds. The mean blood:liver:fat tissue ratios were 1:22:359, which is of the same order of magnitude as for 2,29,4,49,5,59-hexachlorobiphenyl (Wyss et al., 1986) or 2,29,39,4,49,5,59-heptachlorobiphenyl (Koss et al., 1993). The higher levels of PCB accumulation in fat tissue may reflect the relatively great lipophilicity of the congeners, as well as the markedly longer half-life of PCBs in adipose tissue relative to the liver (calculated half-lives for total PCBs in the two tissues being ;7 weeks and 2 to 3 weeks, respectively) (Tanabe et al., 1981; Dragnev et al., 1994). There are few reports indicating that ortho-substituted congeners cause neurobehavioral changes and affect the learning process. Schantz et al. (1995) reported that perinatal exposure to ortho-substituted PCBs (2,4,49-trichlorobiphenyl, 2,39,4,49,5-pentachlorobiphenyl, and 2,29,4,49,5,59-hexachlorobiphenyl) can result in long-lasting deficits in learning. Recently, Eriksson and Fredriksson (1996) reported that neonatal exposure of mice to lightly chlorinated ortho-substituted PCBs (2,4,49-trichlorobiphenyl, 2,29,5,59-tetrachlorobiphenyl) affect behavior of the offspring, while exposure to more heavily chlorinated mono-ortho-substituted congeners (2,39,4,49,5pentachlorobiphenyl and 2,3,39,4,49,5-hexachlorobiphenyl) had no effects at the same doses. Additional studies are needed to confirm whether ortho-substituted congeners contribute to a greater extent to the neurotoxic effects of PCBs. In summary, these results indicate that (1) only ortho-substituted, noncoplanar PCBs accumulate differentially in selected brain regions at the detection limits; (2) total PCB concentrations were as high as 15 ppm (equivalent to 40 –50 mM), concentrations at which intracellular second messengers are significantly affected in neuronal cultures and brain homogenate preparations in vitro; (3) the tissue profile of congeners reflects the composition of parent mixture qualitatively; and (4) concentrations that altered Ca21 disposition and second messenger systems in vitro are achievable in brain in vivo following repeated exposure.
209
ACKNOWLEDGMENTS Congener-specific analysis of PCBs was performed under a U.S. EPA Contract No. 5D1415NAGX. We thank Drs. Susan Schantz, George Frame, and Dennis Morse for their critical comments on the earlier version of this manuscript.
REFERENCES Brouwer, A., Ahlborg, U. G., Van den Berg, M., Birnbaum, L. S., Boersma, E. R., Bosveld, B., Denison, M. S., Gray, L. E., Hagmar, L., Holene, E., Huisman, M., Jacobson, S. W., Jacobson, J. L., Koopman-Esseboom, C., Koppe, J. G., Kulig, B. M., Morse, D. C., Muckle, G., Peterson, R. E., Sauer, P. J. J., Seegal, R. F., Smit-van Prooije, A. E., Touwen, B. C. L., WeisglasKuperus, N., and Winneke, G. (1995). Functional aspects of developmental toxicity of polyhalogenated aromatic hydrocarbons in experimental animals and human infants. Eur. J. Pharmacol. 293, 1– 40. Bush, B., Murphy, M. J., Connor, S., Snow, J., and Bernard, E. (1985). Improvements in glass capillary gas chromatographic polychlorobiphenyl analysis. J. Chromatogr. Sci. 23, 509 –515. Bush, B., Streeter, R. W., and Sloan, R. J. (1989). Polychlorobiphenyl (PCB) congeners in Striped Bass (Morone saxatilis) from marine and estuarine waters of New York State determined by capillary gas chromatography. Arch. Environ. Contam. Toxicol. 19, 49 – 61. Dewailly, E., Hansen, J. C., Pedersen, H. S., Mulvad, G., Ayotte, P., Weber, J. P., and Lebel, G. (1995). Concentration of PCBs in various tissues from autopsies in Greenland. Organohalogen Compd. 26, 175–180. Dragnev, K. H., Beebe, L. E., Jones, C. R., Fox, S. D., Thomas, P. E., Nims, R. W., and Lubet, R. A. (1994). Subchronic dietary exposure to Aroclor 1254 in rats: Accumulation of PCBs in liver, blood, and adipose tissue and its relationship to induction of various hepatic drug-metabolizing enzymes. Toxicol. Appl. Pharmacol. 125, 111–122. Erickson, M. D. (1986). Analytical Chemistry of PCBs. Butterworth, Boston. Eriksson, P., and Fredriksson, A. (1996). Developmental neurotoxicity of four ortho-substituted polychlorinated biphenyls in the neonatal mouse. Environ. Toxicol. Pharmacol. 1, 155–165. Farber, J. L. (1990). The role of Ca21 in lethal cell injury. Chem. Res. Toxicol. 3, 503–508. Fischbein, A., Wolff, M. S., Lilis, R., Thornton, J., and Selikoff, I. J. (1979). Clinical findings among PCB-exposed workers in a capacitor-manufacturing facility. Ann. NY Acad. Sci. 320, 703–715. Fischer, L. J., Seegal, R. F., Ganey, P. E., Pessah, I. N., and Kodavanti, P. R. S. (1998). Sympossium overview: Toxicity of noncoplanar PCBs. Toxicol. Sci. 41, 49 – 61. Frame, G. M., Cochran, J. W., and Bowadt, S. S. (1996). Complete PCB congener distributions for 17Aroclor mixtures determined by 3 HRGC systems optimized for comprehensive, quantitative, congener-specific analysis. J. High Resolut. Chromatogr. 19, 657– 668. Gospe, Jr., S. M., and Calaban, M. J. (1988). Central nervous system distribution of inhaled toluene. Fundam. Appl. Toxicol. 11, 540 –545. Hansen, L. G. (1987). Food chain modification of the composition and toxicity of polychlorinated biphenyl (PCB) residues. Rev. Environ. Toxicol. 3, 149 – 212. Hansen, L. G. (1998). Stepping backward to improve assessment of PCB congener toxicities. Environ. Health Perspect. 106(Suppl. 1), 171–189. Kafafi, S. A., Afeefy, H. Y., Ali, A. H., Said, H. K., and Kafafi, A. G. (1993). Binding of polychlorinated biphenyls to the aryl hydrocarbon receptor. Environ. Health Perspect. 101, 422– 428. Kodavanti, P. R. S., 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
210
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changes in intracellular Ca21 buffering and protein kinase C activity in the absence of changes in tyrosine hydroxylase. Toxicol. Appl. Pharmacol. 153, 186 –198. Kodavanti, P. R. S., Shafer, T. J., Ward, T. R., Mundy, W. R., Freudenrich, T., Harry, G. J., and Tilson, H. A. (1994). Differential effects of PCB congeners on phosphoinositide hydrolysis and protein kinase C translocation in rat cerebellar granule cells. Brain Res. 662, 75– 82. Kodavanti, P. R. S., Shin, D., Tilson, H. A., and Harry, G. J. (1993). Comparative effects of two polychlorinated biphenyl congeners on calcium homeostasis in rat cerebellar granule cells. Toxicol. Appl. Pharmacol. 123, 97–106. Kodavanti, P. R. S., and Tilson, H. A. (1997). Structure–activity relationships of potentially neurotoxic PCB congeners in the rat. Neurotoxicology 18, 423– 438. Kodavanti, P. R. S., Ward, T. R., McKinney, J. D., and Tilson, H. A. (1995). Increased [3H]phorbol ester binding in rat cerebellar granule cells by PCB mixtures and congeners: Structure–activity relationships. Toxicol. Appl. Pharmacol. 130, 140 –148. Kodavanti, P. R. S., Ward, T. R., McKinney, J. D., and Tilson, H. A. (1996). Inhibition of microsomal and mitochondrial Ca21-sequestration in rat cerebellum by polychlorinated biphenyl mixtures and congeners: Structure– activity relationships. Arch. Toxicol. 70, 150 –157. Koss, G., Meyer-Rogge, D., Seubert, S., Seubert, A., and Losekam, M. (1993). 2,29,39,4,49,5,59-heptachlorobiphenyl (PCB 180)— on its toxicokinetics, biotransformation and porphyrinogenic action in female rats. Arch. Toxicol. 67, 651– 654. Kuratsune, M., and Shapiro, R. E. (1984). PCB poisoning in Japan and Taiwan. Prog. Clin. Biol. Res. 137, 1–155. Kuwabara, K., Yakushiji, T., Watanabe, I., Yoshida, S., Koyama, K., Kunita, N., and Hara, I. (1978). Relationship between breast feeding and PCB residues in blood of the children whose mothers were occupationally exposed to PCBs. Int. Arch. Occup. Environ. Health 41, 189 –197. Masuda, Y., Kagawa, H., Tokudome, S., and Kuratsune, M. (1979). Transfer of various polychlorinated biphenyls to the fetuses and offspring of mice. Food Cosmet. Toxicol. 17, 623– 627. Masuda, Y., Kuroki, H., Yamaryo, T., Haraguchi, K., Kuratsune, M., and Hsu, S. (1982). Comparison of causal agents in Taiwan and Fukuoka PCB poisonings. Chemosphere 11, 199 –206. Mattson, M. P. (1991). Evidence for the involvement of protein kinase C in neurodegenerative changes in cultured human cortical neurons. Exp. Neurol. 112, 95–103. McKinney, J, D., and Waller, C. L. (1994). Polychlorinated biphenyls as hormonally active structural analogs. Environ. Health Perspect. 102, 290 – 297. Ness, D. K., Schantz, S. L., and Hansen, L. G. (1994). PCB congeners in the rat brain: Selective accumulation and lack of regionalization. J. Toxicol. Environ. Health 43, 453– 468. Patterson, Jr., D. G., Todd, G. D., Turner, W. E., Maggio, V., Alexander, L. R., and Needham, L. L. (1994). Levels of non-ortho-substituted (coplanar), mono- and di-ortho-substituted polychlorinated biphenyls, dibenzo-p-dioxins, and dibenzofurans in human serum and adipose tissue. Environ. Health Perspect. 102, 195–204. Safe, S. H. (1994). Polychlorinated biphenyls (PCBs): Environmental impact,
biochemical and toxic responses, and implications for risk assessment. Crit. Rev. Toxicol. 24, 87–149. Schantz, S. L., Moshtaghian, J., and Ness, D. K. (1995). Spatial learning deficits in adult rats exposed to ortho-substituted PCB congeners during gestation and lactation. Fundam. Appl. Toxicol. 26, 117–126. Schecter, A., Stanley, J., Boggess, K., Masuda, Y., Mes, J., Wolff, M., Furst, P., Furst, C., Wilson-Yang, K., and Chisholm, B. (1994). Polychlorinated biphenyl levels in the tissues of exposed and non-exposed humans. Environ. Health Perspect. 102, 149 –158. Schultz, D. E., Petrick, G., and Duinker, J. C. (1989). Complete characterization of polychlorinated biphenyl congeners in commercial Aroclor and Clophen mixtures by multidimentional gas chromatography-electron capture detection. Environ. Sci. Technol. 23, 852– 859. Seegal, R. F., Bush, B., and Brosch, K. O. (1991). Sub-chronic exposure of the adult rat to Aroclor 1254 yields regionally-specific changes in central dopaminergic function. Neurotoxicology 12, 55– 66. Seegal, R. F., and Schantz, S. L. (1994). Neurochemical and behavioral sequelae of exposure to dioxins and PCBs. In Dioxins and Health (A. Schecter, Ed.), pp. 409 – 447. Plenum Press, New York. Seegal, R. F., and Shain, W. (1992). Neurotoxicity of polychlorinated biphenyls: The role of ortho-substituted congeners in altering neurochemical function. In The Vulnerable Brain and Environmental Risks, Vol. 2. Toxins in Food (R. L. Isaacson and K. F. Jensen, Eds.), pp. 169 –195. Plenum Press, New York. Shafer, T. J., Mundy, W. R., Tilson, H. A., and Kodavanti, P. R. S. (1996). Disruption of inositol phosphate accumulation in cerebellar granule cells by polychlorinated biphenyls: A consequence of altered Ca21 homeostasis. Toxicol. Appl. Pharmacol. 141, 448 – 455. Shain, W., Overmann, S. R., Wilson, L. R., Kostas, J., and Bush, B. (1986). A congener analysis of polychlorinated biphenyls accumulating in rat pups after perinatal exposure. Arch. Environ. Contam. Toxicol. 15, 687–707. Smith, A., Schloemer, J., Lowry, L., Smallwood, A., Ligo, R., Tanaka, S., Stringer, W., Jones, M., Hervin, R., Glueck, C. (1982). Metabolic and health consequences of occupational exposure to polychlorinated biphenyls. Br. J. Ind. Med. 39, 361–369. Tanabe, S., Nakagawa, Y., and Tatsukawa, R. (1981). Absorption efficiency and biological half-life of individual chlorobiphenyls in rats treated with Kanechlor products. Agric. Biol. Chem. 45, 717–726. Tilson, H. A., and Kodavanti, P. R. S. (1997). Neurochemical effects of polychlorinated biphenyls: An overview and identification of research needs. Neurotoxicology 18, 727–744. U.S. EPA (United States Environmental Protection Agency) (1993). Workshop Report on Developmental Neurotoxic Effects Associated with Exposure to PCBs. Prepared by Eastern Research Group, Inc., Lexington, MA. Prepared for Risk Assessment Forum, EPA/630/R-92/004. World Health Organization (1993). Environmental Health Criteria 140: Polychlorinated Biphenyls and Terphenyls, 2nd ed. Prepared by S. Dobson and G.J. van Esch. International Programme on Chemical Safety, Geneva, Switzerland. Wyss, P. A., Muhlebach, S., and Bickel, M. M. (1986). Long-term pharmacokinetics of 2,29,4,49,5,59-hexachlorobiphenyl (6-CB) in rats with constant adipose tissue mass. Drug Metab. Dispos. 14, 361–365.
153, 199 –210 (1998)
TO988534
Congener-Specific Distribution of Polychlorinated Biphenyls in Brain Regions, Blood, Liver, and Fat of Adult Rats Following Repeated Exposure to Aroclor 12541,2 Prasada Rao S. Kodavanti,* Thomas R. Ward,* Ethel C. Derr-Yellin,* William R. Mundy,* Ann C. Casey,‡ Brian Bush,†,‡ and Hugh A. Tilson* *Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, †Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201, and ‡School of Public Health, University at Albany, Albany, New York 12201– 0509 Received December 3, 1997; accepted July 17, 1998
Congener-Specific Distribution of Polychlorinated Biphenyls in Brain Regions, Blood, Liver, and Fat of Adult Rats Following Repeated Exposure to Aroclor 1254. Kodavanti, P. R. S., Ward, T. R., Derr-Yellin, E. C., Mundy, W. R., Casey, A. C., Bush, B., and Tilson, H. A. (1998). Toxicol. Appl. Pharmacol. 153, 199 –210. Our previous in vitro studies with both isolated organelles and primary neuronal cell cultures found that intracellular signal transduction can be perturbed by some noncoplanar PCBs at exposure levels of <10 mM. However, it is not clear whether such concentrations are achievable in brain in vivo. Also, the pattern of congener disposition and quantities of the PCB accumulation in tissues of animals exposed to commercial PCB mixtures is not well studied. In the present study, we have conducted PCB congenerspecific analysis in different brain regions, liver, blood, and fat of adult male Long–Evans rats dosed orally with Aroclor 1254 (0 or 30 mg/kg/day; once per day, 5 days/week for 4 weeks) in corn oil. Twenty-four hours after the last dose, rats were euthanized, and the brains were removed and dissected to obtain cerebellum, frontal cortex, and striatum. Liver, blood, and fat samples were also collected at the same time. Congener-specific analysis of PCBs was performed by high-resolution gas chromatography with electron capture detection. While PCB concentrations in control rat brain regions were less than 0.02 ppm, total PCB congeners in treated animals accumulated to much higher levels. Total levels in the frontal cortex, cerebellum, and striatum were 15.1 6 0.3, 13.1 6 1.7, and 8.2 6 2.6 ppm, respectively. The levels of PCBs in the fat, liver, and blood were 0.041, 0.002, and 0.001 ppm in control rats and 552, 38.3, and 1.6 ppm in treated rats, respectively. In addition to the differential total uptake between tissues, there was differential accumulation of PCBs with respect to the number of chlorines. In all the tissues, the more lightly chlorinated (tetra- and 1
Presented at the Annual Meeting of Society For Neuroscience, Washington, D.C., November 1996. (Soc. Neurosci. Abstr. 22, 1910, 1996). 2 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.
penta-) congeners accumulated less than their respective proportions in the parent Aroclor 1254 mixture. On the other hand, heavily chlorinated (hexa- to nona-) congeners accumulated more than the proportion of these congeners found in Aroclor 1254 mixture. This shift toward accumulation of heavily chlorinated congeners seems to be more pronounced in the brain than liver and fat. Predominant congeners (5–32% of total PCBs) detected in different brain regions, blood, liver, and fat are: 2,3,3*,4*,5,6- (no. 163) 1 2,2*,3,4,4*,5- (no. 138) (coeluted); 2,2’,4,4*,5,5*- (no. 153) 1 2,2*,3,3*,4,6*- (no. 132) (coeluted); 2,3,3*,4,4*,5- (no. 156) 1 2,2*,3,3*,4,4*,6- (no. 171) (coeluted); 2,3*,4,4*,5- (no. 118); 2,2*,4,4*,5-(no. 99); and 2,3,3*,4,4*- (no. 105). These congeners together accounted for about two thirds of the total PCB load in brain. All these predominant congeners are ortho-substituted and therefore are noncoplanar in nature. The total PCB concentrations accumulated in brain were as high as 50 mM (based on average molecular weight of 326.4 for Aroclor 1254) and, at these concentrations, intracellular second messengers were significantly affected in neuronal cultures and brain homogenate preparations in vitro. These results indicate that concentrations that altered Ca21 disposition and second messenger systems in vitro are achievable in brain in vivo following repeated exposure. © 1998 Academic Press
Polychlorinated biphenyls (PCBs) are persistent environmental contaminants and have become distributed throughout the entire ecosystem. Human populations have been exposed to these chemicals by both chronic and acute exposures. Chronic exposures have occurred as low level environmental exposures as well as high level occupational exposures (Kuwabara et al., 1978; Fischbein et al., 1979; Smith et al., 1982). Acute exposures have occurred through accidental ingestion (Masuda et al., 1982; Kuratsune and Shapiro, 1984). PCBs, which exist as 209 possible congeners, have been reported to cause a variety of effects including immunologic, teratogenic, reproductive, carcinogenic, and neurological effects (Kafafi et al., 1993; Seegal and Schantz, 1994). PCBs are not only a potential health threat to adults, but also to developing fetuses and
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infants, because they cross the human placenta (Masuda et al., 1979) and are found in human milk (U.S. EPA, 1993; WHO, 1993). The effects elicited by PCBs seem to be dependent both on the structural characteristics of the congener (McKinney and Waller, 1994; Fischer et al., 1998) and on the time period of exposure (WHO, 1993). Current risk assessment for PCBs has been based on information from commercial PCB mixtures. Congener-specific actions have long been known (Hansen, 1987, Hansen, 1998; Safe, 1994) and workshops conducted by U.S. EPA and European Environmental Research Organization confirmed that congener-specific information is needed to better evaluate the risk potential of these compounds (U.S. EPA, 1993; Brouwer et al., 1995). Our laboratory explored the cellular mechanism(s) for the neurological effects of PCBs in primary neuronal cell cultures as well as brain homogenates. Our in vitro studies indicated that calcium homeostasis and other second messenger systems including inositol phosphate and protein kinase C (PKC) translocation are sensitive to noncoplanar PCBs at concentrations of #30 mM where cytotoxicity as measured by LDH leakage was not detected (Kodavanti and Tilson, 1997). These studies have been extended to in vivo in adult rats following repeated exposure to a commercial PCB mixture. The changes in second messenger systems seen in vivo (Kodavanti et al., 1998) are similar to those reported in vitro in neuronal preparations. In this study, we conducted congener-specific analysis of PCBs in order to determine (1) if concentrations of PCBs that altered second messenger systems in vitro are achievable in vivo, (2) if the profile of congeners in brain and other tissues reflect the composition of the parent mixture, and (3) if there is a differential PCB accumulation in different brain regions. MATERIALS AND METHODS Chemicals. A commercial PCB mixture, Aroclor 1254, (purity . 99%; lot no. 6024) was purchased from AccuStandard Inc. (New Haven, CT) for dosing rats. Aroclors 1221, 1016, 1254, and 1260 standards were obtained from Ultra Scientific (North Kingstown, RI). Florisil was obtained from U.S. Silica (Pittsburgh, PA). The solvents used in extraction of samples were nanograde from Mallinckrodt, Inc. (St. Louis, MO). All other chemicals used in the assays were obtained from commercial sources. Animals. Adult male (60 days; 250 –270 g) Long–Evans hooded rats were obtained from Charles River Laboratory (Raleigh, NC) and housed two per cage in American Association for Accreditation of Laboratory Animal Care (AAALAC) approved animal facilities. All experiments were approved in advance by the National Health and Environmental Effects Research Laboratory animal care committee of the U.S. EPA. Food and water were provided ad libitum. Temperature was maintained at 21 6 2°C and relative humidity was maintained at 50 6 10% with a 12-h light/dark cycle (light 7:00 to 19:00 h). In vivo dosing. After 1 week of acclimation, rats were dosed by oral gavage with Aroclor 1254 in corn oil (2 ml/kg). The selected dosages were 0 or 30 mg/kg/day. The rats (four rats per group) were dosed five times per week for 4 weeks. The dosing time was always between 8:00 and 10:00 a.m. The animals were weighed three times per week. Twenty-four hours after the last dosage, rats were euthanized and blood was collected. Liver slices (about 1 g) were collected from the main lobes and fat was collected from the abdomen. Different brain regions (cerebellum, frontal cortex, and striatum) were dis-
sected as described previosly (see companion paper, Kodavanti et al., 1998). All the samples were frozen on dry ice, and stored at 280°C until congenerspecific analysis of PCBs. Neurochemical measurements were made in the same batch of animals (see companion paper, Kodavanti et al., 1998) Extraction of PCB congeners in tissues and blood. Congener-specific analysis of PCBs was performed using a high-resolution gas chromatography with electron capture detection (Bush et al., 1985, 1989). Briefly, tissue samples were mixed with anhydrous sodium sulfate to remove water. The tissue sample and sodium sulfate were then ground together with 20 ml of hexane:acetone (1:1) in a SDT tissumizer (Tekmar, Cincinnati, OH) for 1 min. The supernatant hexane was decanted into a Kuderna-Danish evaporator. The extraction was repeated twice more and the combined extracts were concentrated to approximately 2 ml on a steam bath. For extraction of PCBs from whole blood, an equal amount of methanol was added and the tube contents were shaken vigorously. The mixture was left for 15 min in order to break down the proteins and eliminate any blood borne pathogens. To this mixture, 15 ml of diethylether:hexane (1:1) was added and the tubes were shaken again. When the layers separated, the upper layer was pipetted into a Kuderna-Danish evaporator. The extraction was repeated two more times and the combined extracts were concentrated to approximately 1 ml. A 0.1-ml aliquot was used for lipid analysis and the rest of the sample was processed as per tissues.
TABLE 1 Congener-Specific Analysis of PCBs in Water and Rat Chow Used in Aroclor 1254 Study
Water/chow Water
Rat chow
PCB congener Total PCBs 2, 49 and 2, 3 2, 29, 4, 49 and 2, 29, 4, 5 Total PCBs 2, 4 and 2, 5 2, 49 and 2, 3 2, 29, 5, 59 2, 29, 3, 59 2, 29, 3, 5, 69 and 2, 4, 49, 5 2, 39, 49, 5 2, 39, 4, 49 2, 29, 4, 5, 59 2, 29, 4, 49, 5 2, 29, 39, 4, 5 2, 29, 3, 4, 59 3, 39, 4, 49 and 2, 3, 39, 49, 6 2, 29, 3, 39, 5, 69 2, 29, 3, 49, 59, 6 2, 39, 4, 49, 5 2, 29, 3, 49, 5, 59 2, 29, 4, 49, 5, 59 and 2, 29, 3, 39, 4, 69 2, 3, 39, 4, 49 2, 29, 3, 4, 49, 59 2, 29, 3, 39, 4, 5, 6, 69 DDE Mirex
IUPAC no.
8 5 47 48 7 9 8 5 52 44 94 74 70 66 101 99 97 87 77 110 135 149 118 146 153 132 105 138 200
Concentration (ppb; ng/g or ml) 1.422 0.986 0.436 4.730 0.457 0.227 0.258 0.137 0.140 0.317 0.294 0.420 0.315 0.108 0.076 0.380 0.155 0.124 0.221 0.042 0.205 0.080 0.479 0.295 0.253 0.757
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TABLE 2 Different PCB Congeners Based on the Number of Chlorines Detected in Different Brain Regions, Blood, Liver, and Fat of Adult Rats Dosed with Aroclor 1254 PCB congeners (ppb; ng/g tissue or ml blood) Tissue
Mono, di- and tri-
Tetra-
Penta-
Hexa-
Hepta-
Octa- and nona
Aroclor 1254 Brain Frontal cortex Cerebellum Striatum Blood Liver Fat
(2.1)
(19.1)
(49.6)
(25.9)
(2.9)
(0.5)
6 (,1) 2 (,1) 3 (,1) 0 (0) 0 (0) 0 (0)
542 (4) 577 (4) 223 (3) 47 (3) 180 (,1) 30321 (6)
3964 (26) 3652 (28) 2346 (28) 540 (35) 12850 (34) 249760 (45)
7970 (53) 7044 (54) 4464 (54) 746 (48) 21961 (57) 223275 (41)
2304 (15) 1584 (12) 1030 (13) 153 (10) 2116 (6) 33779 (6)
330 (2) 259 (2) 173 (2) 61 (4) 1166 (3) 14416 (2)
Note. Mean values are reported (n 5 3). The numbers in parenthesis indicate the percentage of total PCBs in that tissue. Dosage of Aroclor 1254 was 30 mg/kg/day; 5 days/week for 4 weeks.
PCB congener-specific analysis. The concentrated solution was then transferred quantitatively to a 1 3 15 cm chromatography column containing 10 g of calibrated 4% deactivated Florisil with a 2-g layer of sodium sulfate on top of the Florisil. The column was eluted with 50 ml of hexane containing PCBs, concentrated to 1 ml using a Kuderna-Danish evaporator, and pipetted to a 1 ml Wheaton GC vial for analysis. The samples were analyzed on a Hewlett-Packard 5890 gas chromatograph equipped with a Ni-63 electron capture detector with a fused silica column with cross-linked 5% phenylmethylsilicone coating (0.33 mm film thickness, 0.25 mm id). The oven temperature was held at 100°C for 2 min, and then raised up to 160°C at 10°C/min, then 1°C/min to 190°C, and then 2°C/min to a final temperature of 270°C. The temperature was held at 270°C for 10 min until all congeners were eluted. Approximately 113 PCB congeners were identified and verified using a separate Apiezon L capillary column (Bush et al., 1985). The microprocessor of the gas chromatograph was calibrated with a solution of Aroclors 1221, 1016, 1254, and 1260 (200 ng/ml of each) fortified with hexachlorobenzene (HCB), 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), and mirex at 5, 10, and 10 ng/ml, respectively, and 3,39,4,49-tetrachlorobiphenyl at 20 ng/ml. This mixture has been previously fully characterized (Bush et al., 1985; Schultz et al., 1989). Data analysis was carried out with an IBM compatible personal computer using FILESERVER and entered into LOTUS 123 (Lotus Development Corp., Cambridge, MA). Quality control for the analysis was ensured by running replicates of a sample in every batch of 10 samples that were analyzed to ensure the consistency of sample extraction. Replicate samples of the first set of fat, liver, and brain of each group were extracted and analyzed on a GC with a Hewlett Packard Ultra II and an Apiezon L column. The Ultra II is used as a primary column for quantitation and Apiezon L is used to confirm peak identities. The samples were spiked with DDE to standardize retention times and check percent recovery of the extraction process. The average minimum detectable level (MDL) (p , 0.05 that the reported level is zero) (Type 1 error) for a sample weighing 1 g was 0.02 ng/g (U.S. EPA, 1984). It should be noted that even at one quarter of this value, the chances for the presence of that congener is 87% (Type II error). The results of PCB analysis were expressed as ng or or mg PCB/g wet weight tissue or ml blood.
RESULTS
respectively (Table 1). The trace amounts of PCBs in rat chow and water are typical of the background levels reported previously (Shain et al., 1986) and will not interfere with the interpretation of results from the present experiment. The accumulation of PCBs in the control rat tissues was very low compared to the treated animals. The total PCB accumulation in the brains of control animals was less than 0.02 ppm. Other tissues also had relatively low levels, with the fat containing the highest level (0.041 ppm). In all control tissues, total PCBs and individual congeners were less than 1% of treated values. Different PCB Congeners in the Commercial Mixture, Aroclor 1254 (Lot No. 6024) The distribution of congeners in the Aroclor 1254 is shown in Tables 2, 4, and 5. In agreement with published reports TABLE 3 Toxicokinetic Distribution of PCB Congeners in Adult Rats Dosed with Aroclor 1254 Total PCBs (ppm; mg/g tissue or ml blood) Control
Brain Frontal cortex Cerebellum Striatum Blood Liver Fat
0.019a 0.004 0.000 0.001 0.002 0.041
Aroclor 1254
15.12 6 0.31b 13.13 6 1.68 8.24 6 2.57 1.55 6 0.01 38.27 6 8.22 551.55 6 41.06
Note. Dosage of Aroclor 1254 was 30 mg/kg/day; 5 days/week for 4 weeks. Since controls give information about background levels of exposure, which is minimal, only one animal was analyzed. b For Aroclor 1254-treated animals, three rats were analyzed and the values reported are means 6 SE of three rats. a
The rat chow and water given to control and exposed rats contained low levels of a number of PCB congeners (Table 1). Total PCBs in water and rat chow were 1.4 and 4.7 ppb,
Tissue
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TABLE 4 Congener-Specific Analysis of PCBs in Different Regions of Brain of Rats Treated with Aroclor 1254 Congener Total PCB congeners 3, 39, 4, 491 2, 3, 39, 49, 6 2, 29, 4, 5, 59 2, 39, 4, 49, 5 2, 29, 3, 39, 49, 5, 61 2, 29, 3, 4, 49, 59 2, 39, 4, 49 2, 29, 3, 59, 6 2, 39, 49, 5 2, 29, 4, 49, 5, 591 2, 29, 3, 39, 4, 69 2, 3, 39, 4, 49 2, 29, 4, 49, 5 2, 29, 39, 4, 5 2, 29, 5, 59 2, 29, 3, 49, 59, 6 2, 29, 3, 4, 59 DDE 2, 3, 4, 491 29, 3, 39, 4 2, 29, 3, 39, 4, 491 2, 39, 4, 49, 5, 59 2, 29, 3, 59 2, 29, 3, 5, 691 2, 4, 49, 5 2, 29, 3, 39, 4, 59 2, 3, 39, 4, 49, 6 2, 29, 3, 39, 4, 49, 61 2, 3, 39, 4, 49, 5 2, 29, 3, 39, 4, 5 2, 29, 3, 49, 5, 6 2, 29, 3, 39, 5, 69 2, 29, 4, 59 2, 29, 3, 4, 5, 591 2, 29, 3, 39, 5, 6, 69 2, 29, 3, 4, 49, 5, 59 2, 29, 3, 39, 4, 49, 51 2, 3, 39, 4, 49, 5, 6 2, 29, 3, 49, 5, 59 2, 3, 49, 6 2, 29, 3, 39, 4, 6, 69 2 2, 29, 3, 39, 6, 69 2, 29, 3, 5, 59, 61 2, 29, 3, 39, 4 2, 29, 3, 39, 5, 6 2, 49, 5 2, 29, 3, 39, 4, 59, 6, 69 2, 3, 39, 61 2, 29, 3, 491 3, 4, 49
IUPAC no.
771 110 101 118 1631 138 661 95 70 1531 132 105 99 97 52 149 87 601 56 1281 167 44 941 74 130 158 1711 156 129 147 135 49 1411 179 180 1701 190 146 64 176 1 136 1511 82 134 31 201 591 421 37
Aroclor 1254
Cerebellum
Frontal cortex
Striatum
100.00 13.21
100.00 0.07
100.00 0.07
100.00 0.02
9.94 9.81 7.87
0.43 12.30 19.21
0.40 11.63 18.43
0.40 12.94 19.42
5.53
1.69
1.17
1.96
5.11 4.52
1.70 17.24
1.59 16.57
0.00 17.10
4.38 4.32 3.42 2.86 2.80 2.57 2.34 2.27
4.93 8.93 0.10 0.00 0.41 0.49 0.68 0.49
4.79 8.31 0.09 0.00 0.27 0.51 0.64 0.47
4.81 9.15 0.10 0.00 0.20 0.41 0.65 0.46
2.10
0.73
0.70
0.76
1.46 1.26
0.00 0.99
0.00 0.66
0.00 0.88
1.22 1.16 1.16
0.94 0.32 10.66
0.93 0.47 11.12
0.93 1.16 10.38
1.02 0.86 0.86 0.74 0.69
0.30 0.03 0.00 0.00 0.02
0.38 0.02 0.01 0.00 0.02
0.30 0.14 0.12 0.00 0.05
0.68 0.65
1.98 3.39
2.39 3.43
2.89 3.19
0.65 0.60 0.56 0.54 0.50 0.49
2.42 0.00 3.31 0.00 0.00 0.01
2.40 0.00 6.20 0.00 0.00 0.04
2.33 0.00 3.16 0.00 0.00 0.00
0.49 0.47 0.38 0.30
2.08 0.00 1.53 0.00
2.12 0.00 1.57 0.00
2.00 0.00 1.68 0.00
Note. Dosage of Aroclor 1254 was 30 mg/kg/day; 5 days/week for 4 weeks. All values are % of total: total PCBs in cerebellum 5 13, 128 ng/g; frontal cortex 5 15, 118 ng/g; and striatum 5 8239 ng/g; n 5 3.
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TABLE 4—Continued Congener 2, 29, 3, 39, 4, 5, 69 2, 29, 3, 39 2, 29, 4, 491 2, 29, 4, 5 4 2, 29, 3, 49, 5, 59, 61 2, 29, 3, 4, 49, 5, 69 2, 29, 3, 4, 49, 59, 6 2, 29, 5 2, 49,1 2, 3 2, 29, 3, 39, 49, 5, 6 2, 29, 31 2, 49, 6 29, 3, 4 2, 291 2, 6 4, 491 2, 29, 4 2, 29, 3, 6 2, 3, 49 2, 29, 3, 39, 4, 5, 59 2, 39 2, 29, 3, 4, 5, 59, 6 2, 29, 3, 39, 4, 5, 69 2, 29, 3, 4, 49, 5, 59, 61 2, 29, 3, 39, 4, 49, 5, 69 2, 29, 3, 39, 4, 49, 5, 59 2, 29, 3, 39, 4, 5, 59, 69 2, 39, 5 2, 41 2, 5 HCB 2, 3, 61 2, 39, 6 2, 29, 6 2, 39, 4 2, 4, 49 Mirex 2, 29, 3, 39, 4, 49, 5, 6 2, 29, 3, 39, 4, 49, 5, 59, 6
IUPAC no. 174 40 471 48 3 1871 182 183 18 81 5 177 161 32 33 41 10 151 17 45 22 172 6 185 200 2031 196 194 199 26 71 9 241 27 19 25 28 195 206
Aroclor 1254
Cerebellum
Frontal cortex
Striatum
0.28 0.28 0.24
0.06 0.00 0.09
0.06 0.00 0.08
0.00 0.00 0.06
0.23 0.19
0.00 0.71
0.00 0.71
0.00 0.73
0.19 0.18 0.16
0.56 0.00 0.00
0.56 0.00 0.00
0.52 0.00 0.00
0.13 0.10
0.51 0.00
0.50 0.00
0.54 0.00
0.09 0.07
0.00 0.00
0.00 0.00
0.00 0.00
0.07
0.00
0.00
0.00
0.06 0.05 0.05 0.03 0.03 0.03 0.02
0.00 0.00 0.31 0.00 0.54 0.03 0.16
0.00 0.00 0.31 0.00 0.35 0.06 0.15
0.00 0.00 0.25 0.00 0.54 0.07 0.11
0.02 0.02 0.01 0.01
0.10 0.10 0.00 0.00
0.05 0.18 0.00 0.02
0.09 0.15 0.00 0.03
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.01 0.00 0.05 0.08
0.00 0.00 0.01 0.00 0.09 0.08
0.00 0.00 0.00 0.00 0.00 0.00
(Bush et al., 1985; Erickson, 1986; Schultz et al., 1989), Aroclor 1254 contained more ortho-substituted (noncoplanar) PCB congeners than non-ortho-substituted (coplanar) congeners. By our procedure, di-to-tetra-ortho-substituted PCBs (nondioxin like) accounted for 54.5% while mono-ortho PCBs (mixed type) were 45% of total PCBs in the Aroclor 1254 mixture. The only possible non-ortho (dioxin-like) congener detected was 3,39,4,49-tetrachlorobiphenyl (no. 77), which coeluted with 2,3,39,49,6-pentachlorobiphenyl (no. 110) and these two congeners together accounted for 13.2% of the total PCBs. Frame et al. (1996) reported that the coplanar PCB congeners (nos. 77, 126, and 169) were less than 1% of total PCBs in two different Aroclor 1254 mixtures. In this study, a special analysis with carbon separation of coplanar PCBs was not con-
ducted on these samples for reasons of cost. However, a separate analysis of this batch of Aroclor 1254 (lot no. 6024) by the Department of Chemistry at University of Kiel, Germany indicated that coplanar non-ortho substituted PCBs (nos. 77, 126, and 169) were 3% of total PCBs (Kodavanti et al., unpublished data). The variations in the composition of these congeners in Aroclor 1254 might be due to differences in the manufacturing process of these mixtures. PCB congenerspecific analysis also revealed that pentachlorobiphenyls were the highest (49.6% of total) followed by hexachlorobiphenyls (25.9% of total) and tetrachlorobiphenyls (19.1% of total). All other PCB congeners were less than 6% of total (Table 2). Of the 88 congeners or pairs (some coeluted), 12 congeners with IUPAC numbers of 77 1 110 (coeluted), 101, 118, 138 1 163
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KODAVANTI ET AL.
(coeluted), 66, 70, 153 1 132 (coeluted), 105, and 99 accounted for over 60% of the total mixture and this is in agreement with a recent report by Frame et al. (1996). DDE was the only major contaminant detected, which was 2.34% of the total mixture. Distribution of PCB Congeners in Different Brain Regions While PCB concentration in control rat brain regions was less than 20 ppb, total PCB congeners in exposed animals accumulated to ppm levels (Table 3). Total PCB congeners (means 6 SE) were greater in frontal cortex (15.1 6 0.3 ppm) and cerebellum (13.1 6 1.7 ppm) compared to striatum (8.24 6 2.57 ppm), suggesting marginal differences in the accumulation of PCBs in brain regions. Distribution of PCBs by number of chlorines in the Aroclor 1254 mixture and the various tissues is shown in Table 2. As expected, the percentage of the various congeners in Aroclor 1254 exhibited a bell-shaped curve with the peak at pentachlorobiphenyls (49.6% total). The distribution of PCBs by number of chlorines in brain is also bell shaped, but the peak was at hexachlorobiphenyls. There was a shift toward accumulation of more heavily chlorinated congeners in all three brain regions. The congeners with five or more chlorines accounted for 78.9% of the total in the Aroclor 1254 mixture while they were approximately 97% in the brain. The proportion of pentachlorobiphenyls was 49.6% in Aroclor 1254 while the accumulation of pentachlorobiphenyls was much lower (26 –28% of total) in brain regions. Although the total level of PCB congeners was slightly lower in the striatum, the ratios of congeners (by weight) among brain regions remained constant (Table 2). Predominant congeners detected in different brain regions were: 2,3,39,49,5,6- (no. 163) 1 2,29,3,4,49,5- (no. 138) (coeluted); 2,29,4,49,5,59- (no. 153) 1 2,29,3,39,4,69- (no. 132) (coeluted); 2,3,39,4,49,5- (no. 156) 1 2,29,3,39,4,49,6- (no. 171) (coeluted); 2,39,4,49,5- (no. 118), 2,3,39,4,49- (no. 105), and 2,29,4,49,5-(no. 99) (Table 4; Fig. 1). Total PCB concentrations observed in brain were as high as 15 ppm, equivalent to 40 to 50 mM based on average molecular weight of 326.4 for Aroclor 1254 mixture (Erickson, 1986) and, at these concentrations, intracellular second messengers were significantly affected in neuronal preparations in vitro (Kodavanti et al., 1993, 1994, 1995). Distribution of PCB Congeners in Blood, Liver, and Fat The levels of total PCBs in the fat were high (552 6 41 ppm). The circulating levels of total PCBs in the blood were 1.55 6 0.01 ppm. Liver accumulated greater amounts of PCBs (38.3 6 8.2 ppm) than any of the brain regions (Table 3). The shift toward accumulation of more heavily chlorinated congeners, as seen in the brain, was also observed in blood, liver, and fat, but to a lesser extent. Blood and liver contained 97 and 99% of the heavier ($penta-) congeners, respectively, compared to 78.9% in the Aroclor 1254 (Table 2). Accumulation of
heavier congeners in fat was slightly less than other tissues accounting for 94% of the total. Predominant congeners detected in blood, liver, and fat were PCBs with IUPAC number of 138 1 163 (coeluted), 118, 99, 105, and 132 1 153 (coeluted) (Table 5; Fig. 2). In addition, PCBs with IUPAC numbers of 156 1 171 were also detected in higher amounts (10% of total) in liver (Table 5; Fig. 2). DISCUSSION
We have previously demonstrated that second messenger systems including inositol phosphate, Ca21 homeostasis, and PKC translocation are preferentially sensitive to noncoplanar PCBs at low micromolar concentrations in neuronal cultures and brain homogenate preparations (Kodavanti and Tilson, 1997; Tilson and Kodavanti, 1997). Higher concentrations ($100 mM) and longer exposure periods (.1 h) were required to see any cytotoxicity. It is known that perturbations in Ca21 homeostasis and sustained activation of PKC are involved in the neurological effects of a variety of chemicals (Farber, 1990; Mattson, 1991). Consistent with our in vitro observations, the data from our in vivo study (Kodavanti et al., 1998) indicate that intracellular Ca21 buffering and PKC activity can be affected in brain regions of rats exposed repeatedly with a commercial PCB mixture, Aroclor 1254. However, it was not known whether concentrations required to see a significant change in vitro are achievable in vivo or the accumulation of congeners reflects the parent mixture or if there is a differential accumulation in brain regions. The present study demonstrated that, following repeated administration of Aroclor 1254, the brain can accumulate PCBs up to 15 ppm, which is equivalent to 40 to 50 mM based on the average molecular weight of 326.4 for Aroclor 1254 mixture (Erickson, 1986). These are the concentrations that have significant effects on second messenger systems in vitro using cell cultures or brain homogenate preparations (Kodavanti et al., 1993, 1994, 1995, 1996; Shafer et al., 1996). Therefore, PCB concentrations which alter calcium buffering and PKC activity in vitro are achievable in vivo. It is noteworthy that most of the congeners accumulated in brain are orthosubstituted and noncoplanar in nature. Similar observations were also made by Seegal and Shain (1992) where several mono- and di-ortho-substituted congeners were detected in rat brain following Aroclor 1254 exposure in diet. Interestingly, Dewailly et al. (1995) reported PCB levels in various tissues from human autopsies in Greenland and mean concentration of total PCBs were 16.9 mg/kg (ppm) in adipose tissue, 12.2 mg/kg in liver, and 2.0 mg/kg in brain lipids. The PCB congeners found at the highest levels in the Aroclor mixture, nos. 138 1 163 (coeluting), 118, 105, and 132 1 153 (coeluting) were also highest in the brain tissue, together accounting for almost 50% of the total. Other congeners nos. 77 1 110, 101, 66 1 95, and 70, which represent 33.8% of the Aroclor 1254 mixture, were less than 2% of the congeners
CONGENER-SPECIFIC DISTRIBUTION OF PCBS IN BRAIN
205
FIG. 1. Congener-specific accumulation of PCBs in different brain regions (frontal cortex, cerebellum, and striatum) of rats treated with Aroclor 1254 (30 mg/kg/day; 5 days/week for 4 weeks). The x-axis shows PCB concentrations in ng/g wet weight tissue (ppb). The y-axis shows IUPAC numbers of PCB congeners in the order of elution from the column. Only mean values (n 5 3) were plotted for clarity.
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KODAVANTI ET AL.
TABLE 5 Congener-Specific Analysis of Polychlorinated Biphenyls in Blood, Liver, and Fat of Rats Treated with Aroclor 1254 Congener Total PCB congeners 3, 39, 4, 49,1 2, 3, 39, 49, 6 2, 29, 4, 5, 59 2, 39, 4, 49, 5 2, 3, 39, 49, 5, 61 2, 29, 3, 4, 49, 59 2, 39, 4, 49 2, 29, 3, 59, 6 2, 39, 49, 5 2, 29, 4, 49, 5, 59, 1 2, 29, 3, 39, 4, 69 2, 3, 39, 4, 49 2, 29, 4, 49, 5 2, 29, 39, 4, 5 2, 29, 5, 59 2, 29, 3, 49, 59, 6 2, 29, 3, 4, 59 DDE 2, 3, 4, 491 29, 3, 39, 4 2, 29, 3, 39, 4, 49,1 2, 39, 4, 49, 5, 59 2, 29, 3, 59 2, 29, 3, 5, 691 2, 4, 49, 5 2, 29, 3, 39, 4, 59 2, 3, 39, 4, 49, 6 2, 29, 3, 39, 4, 49, 61 2, 3, 39, 4, 49, 5 2, 29, 3, 39, 4, 5 2, 29, 3, 49, 5, 6 2, 29, 3, 39, 5, 69 2, 29, 4, 59 2, 29, 3, 4, 5, 591 2, 29, 3, 39, 5, 6, 69 2, 29, 3, 4, 49, 5, 59 2, 29, 3, 39, 4, 49, 51 2, 3, 39, 4, 49, 5, 6 2, 29, 3, 49, 5, 59 2, 3, 49, 6 2, 29, 3, 39, 4, 6, 69 2 2, 29, 3, 39, 6, 69 2, 29, 3, 5, 59, 61 2, 29, 3, 39, 4 2, 29, 3, 39, 5, 6 2, 49, 5 2, 29, 3, 39, 4, 59, 6, 69 2, 3, 39, 61 2, 29, 3, 49 3, 4, 49
IUPAC no.
771 110 101 118 1631 138 661 95 70 1531 132 105 99 97 52 149 87 601 56 1281 167 44 941 74 130 158 1711 156 129 147 135 49 1411 179 180 1701 190 146 64 176 1 136 1511 82 134 31 201 591 421 37
Aroclor 1254
Blood
Liver
Fat
100.00 13.21
100.00 0.49
100.00 0.00
100.00 1.57
9.94 9.81 7.87
0.74 13.63 22.62
0.00 13.03 32.12
2.22 17.80 18.24
5.53
1.53
0.00
3.17
5.11 4.52
0.07 18.59
0.00 18.00
0.32 12.41
4.38 4.32 3.42 2.86 2.80 2.57 2.34 2.27
8.32 10.80 0.20 0.00 0.00 0.20 0.24 0.59
8.04 14.37 0.00 0.00 0.00 0.00 0.25 0.00
7.58 13.65 0.61 0.00 1.14 0.96 1.77 1.11
2.10
1.02
0.00
2.19
1.46 1.26
0.00 1.33
0.00 0.49
0.00 1.84
1.22 1.16 1.16
0.91 2.19 0.61
0.00 0.00 10.05
1.04 3.14 0.41
1.02 0.86 0.86 0.74 0.69
0.00 0.07 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.07 0.11 0.00 0.00 0.00
0.68 0.65
3.80 3.49
2.72 2.47
2.01 1.96
0.65 0.60 0.56 0.54 0.50 0.49
2.51 0.00 0.00 0.00 0.00 0.00
1.67 0.00 0.00 0.00 0.00 0.00
2.21 0.00 0.00 0.00 0.00 0.00
0.49 0.47 0.38 0.30
0.00 0.00 3.31 0.00
0.00 0.00 3.24 0.00
0.00 0.00 2.65 0.00
Note. Dosage of Aroclor 1254 was 30 mg/kg/day; 5 days/week for 4 weeks. All values are % of total: total PCBs in blood 5 1547 ng/g; liver 5 38,273 ng/g; fat 5 551,551 ng/g; n 5 3.
207
CONGENER-SPECIFIC DISTRIBUTION OF PCBS IN BRAIN
TABLE 5—Continued Congener 2, 29, 3, 39, 4, 5, 69 2, 29, 3, 39 2, 29, 4, 491 2, 29, 4, 5 4 2, 29, 3, 49, 5, 59, 61 2, 29, 3, 4, 49, 5, 69 2, 29, 3, 4, 49, 59, 6 2, 29, 5 2, 49,1 2, 3 2, 29, 3, 39, 49, 5, 6 2, 29, 31 2, 49, 6 29, 3, 4 2, 291 2, 6 4, 491 2, 29, 4 2, 29, 3, 6 2, 3, 49 2, 29, 3, 39, 4, 5, 59 2, 39 2, 29, 3, 4, 5, 59, 6 2, 29, 3, 39, 4, 5, 6, 69 2, 29, 3, 4, 49, 5, 59, 61 2, 29, 3, 39, 4, 49, 5, 69 2, 29, 3, 39, 4, 49, 5, 59 2, 29, 3, 39, 4, 5, 59, 69 2, 39, 5 2, 41 2, 5 HCB 2, 3, 61 2, 39, 6 2, 29, 6 2, 39, 4 2, 4, 49 Mirex 2, 29, 3, 39, 4, 49, 5, 6 2, 29, 3, 39, 4, 49, 5, 59, 6
IUPAC no.
Aroclor 1254
Blood
Liver
Fat
0.28 0.28 0.24
0.00 0.00 0.09
0.00 0.00 0.00
0.00 0.00 0.15
0.23 0.19
0.00 0.83
0.00 0.00
0.00 0.47
0.19 0.18 0.16
0.58 0.00 0.00
0.00 0.00 0.00
0.34 0.00 0.00
0.13 0.10
0.55 0.00
0.00 0.00
0.36 0.00
0.09 0.07
0.00 0.00
0.00 0.00
0.00 0.00
0.07
0.00
0.00
0.00
0.06 0.05 0.05 0.03 0.03 0.03 0.02
0.00 0.00 0.00 0.00 0.32 0.02 0.06
0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.30 0.00 0.00
0.02 0.02 0.01 0.01
0.24 0.19 0.00 0.00
0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.10
0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00
174 40 471 48 3 1871 182 183 18 81 5 177 161 32 33 41 10 151 17 45 22 172 6 185 200 2031 196 194 199 26 71 9 241 27 19 25 28 195 206
accumulated in the brain, suggesting that these congeners do not preferentially accumulate in brain, probably due to liver metabolism (Dragnev et al., 1994). On the other hand, no. 99, which was 4.3% of the Aroclor 1254, accounted for about 8 to 9% of the total brain congeners. Similarly, no. 156 1 171, which was only 1.2% of the Aroclor 1254, accounted for about 10 to 11% of the total brain congeners, suggesting preferential accumulation due to less metabolism. In the Greenland study, major congeners detected in human brain were PCBs 99, 105, 118, 138, 153, and 156, which are similar to the ones reported in the present study. PCBs 118, 138, and 153 have been detected in serum samples from U.S. population and Yusho patients (Patterson et al., 1994; Schecter et al., 1994). These same PCB congeners were present at the highest concentra-
tions in frontal cortex, cerebellum, and striatum of Aroclor 1254-dosed rats. Several PCB congeners (IUPAC nos. 99, 118, 138, 153, and 156) that significantly affected intracellular calcium buffering (Kodavanti et al., 1996) and PKC translocation in vitro in neuronal cultures (Kodavanti et al., 1995) are present in the parent Aroclor 1254 mixture. These congeners also accumulated at higher concentrations in brain regions, suggesting a significant role for these congeners in the neurotoxicity of PCBs. Although the congener distribution is similar between the brain regions, the frontal cortex and cerebellum accumulated PCBs to a slightly higher level than did the striatum. The PCB levels for the frontal cortex, cerebellum, and striatum are 15.1,
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KODAVANTI ET AL.
FIG. 2. Congener-specific accumulation of PCBs in blood, liver, and fat tissues of rats treated with Aroclor 1254 (30 mg/kg/day; 5 days/week for 4 weeks). The x-axis shows PCB concentrations in ng/g wet weight tissue or ml blood (ppb). The y-axis shows IUPAC numbers of PCB congeners in the order of elution from the column. Only mean values (n 5 3) were plotted for clarity.
CONGENER-SPECIFIC DISTRIBUTION OF PCBS IN BRAIN
13.1, and 8.2 mg/g wet weight tissue, respectively. Such minor differences in accumulation of PCBs in different brain regions was also reported previously (Seegal et al., 1991). However, Ness et al. (1994) reported no differences in PCB concentrations among brain regions, but the different congeners differed from each other in degree of bioaccumulation in rats developmentally exposed to another Aroclor mixture (Aroclor 1242). The minor differences in the accumulation of PCBs in brain regions, as seen in the present study, cannot be explained simply by differences in lipid content. Gospe and Calaban (1988) reported that total lipid content (as % of wet weight) is similar in all three brain regions (8.9, 8.6, and 8.7 for the frontal cortex, cerebellum, and caudate, respectively). The PCB congener-specific distribution in blood, liver, and fat was qualitatively similar to that of brain regions (Tables 4 and 5). Most of the congeners that accumulated are orthosubstituted and noncoplanar in nature. Also, this study agrees with the typical behavior of highly lipophilic compounds. The mean blood:liver:fat tissue ratios were 1:22:359, which is of the same order of magnitude as for 2,29,4,49,5,59-hexachlorobiphenyl (Wyss et al., 1986) or 2,29,39,4,49,5,59-heptachlorobiphenyl (Koss et al., 1993). The higher levels of PCB accumulation in fat tissue may reflect the relatively great lipophilicity of the congeners, as well as the markedly longer half-life of PCBs in adipose tissue relative to the liver (calculated half-lives for total PCBs in the two tissues being ;7 weeks and 2 to 3 weeks, respectively) (Tanabe et al., 1981; Dragnev et al., 1994). There are few reports indicating that ortho-substituted congeners cause neurobehavioral changes and affect the learning process. Schantz et al. (1995) reported that perinatal exposure to ortho-substituted PCBs (2,4,49-trichlorobiphenyl, 2,39,4,49,5-pentachlorobiphenyl, and 2,29,4,49,5,59-hexachlorobiphenyl) can result in long-lasting deficits in learning. Recently, Eriksson and Fredriksson (1996) reported that neonatal exposure of mice to lightly chlorinated ortho-substituted PCBs (2,4,49-trichlorobiphenyl, 2,29,5,59-tetrachlorobiphenyl) affect behavior of the offspring, while exposure to more heavily chlorinated mono-ortho-substituted congeners (2,39,4,49,5pentachlorobiphenyl and 2,3,39,4,49,5-hexachlorobiphenyl) had no effects at the same doses. Additional studies are needed to confirm whether ortho-substituted congeners contribute to a greater extent to the neurotoxic effects of PCBs. In summary, these results indicate that (1) only ortho-substituted, noncoplanar PCBs accumulate differentially in selected brain regions at the detection limits; (2) total PCB concentrations were as high as 15 ppm (equivalent to 40 –50 mM), concentrations at which intracellular second messengers are significantly affected in neuronal cultures and brain homogenate preparations in vitro; (3) the tissue profile of congeners reflects the composition of parent mixture qualitatively; and (4) concentrations that altered Ca21 disposition and second messenger systems in vitro are achievable in brain in vivo following repeated exposure.
209
ACKNOWLEDGMENTS Congener-specific analysis of PCBs was performed under a U.S. EPA Contract No. 5D1415NAGX. We thank Drs. Susan Schantz, George Frame, and Dennis Morse for their critical comments on the earlier version of this manuscript.
REFERENCES Brouwer, A., Ahlborg, U. G., Van den Berg, M., Birnbaum, L. S., Boersma, E. R., Bosveld, B., Denison, M. S., Gray, L. E., Hagmar, L., Holene, E., Huisman, M., Jacobson, S. W., Jacobson, J. L., Koopman-Esseboom, C., Koppe, J. G., Kulig, B. M., Morse, D. C., Muckle, G., Peterson, R. E., Sauer, P. J. J., Seegal, R. F., Smit-van Prooije, A. E., Touwen, B. C. L., WeisglasKuperus, N., and Winneke, G. (1995). Functional aspects of developmental toxicity of polyhalogenated aromatic hydrocarbons in experimental animals and human infants. Eur. J. Pharmacol. 293, 1– 40. Bush, B., Murphy, M. J., Connor, S., Snow, J., and Bernard, E. (1985). Improvements in glass capillary gas chromatographic polychlorobiphenyl analysis. J. Chromatogr. Sci. 23, 509 –515. Bush, B., Streeter, R. W., and Sloan, R. J. (1989). Polychlorobiphenyl (PCB) congeners in Striped Bass (Morone saxatilis) from marine and estuarine waters of New York State determined by capillary gas chromatography. Arch. Environ. Contam. Toxicol. 19, 49 – 61. Dewailly, E., Hansen, J. C., Pedersen, H. S., Mulvad, G., Ayotte, P., Weber, J. P., and Lebel, G. (1995). Concentration of PCBs in various tissues from autopsies in Greenland. Organohalogen Compd. 26, 175–180. Dragnev, K. H., Beebe, L. E., Jones, C. R., Fox, S. D., Thomas, P. E., Nims, R. W., and Lubet, R. A. (1994). Subchronic dietary exposure to Aroclor 1254 in rats: Accumulation of PCBs in liver, blood, and adipose tissue and its relationship to induction of various hepatic drug-metabolizing enzymes. Toxicol. Appl. Pharmacol. 125, 111–122. Erickson, M. D. (1986). Analytical Chemistry of PCBs. Butterworth, Boston. Eriksson, P., and Fredriksson, A. (1996). Developmental neurotoxicity of four ortho-substituted polychlorinated biphenyls in the neonatal mouse. Environ. Toxicol. Pharmacol. 1, 155–165. Farber, J. L. (1990). The role of Ca21 in lethal cell injury. Chem. Res. Toxicol. 3, 503–508. Fischbein, A., Wolff, M. S., Lilis, R., Thornton, J., and Selikoff, I. J. (1979). Clinical findings among PCB-exposed workers in a capacitor-manufacturing facility. Ann. NY Acad. Sci. 320, 703–715. Fischer, L. J., Seegal, R. F., Ganey, P. E., Pessah, I. N., and Kodavanti, P. R. S. (1998). Sympossium overview: Toxicity of noncoplanar PCBs. Toxicol. Sci. 41, 49 – 61. Frame, G. M., Cochran, J. W., and Bowadt, S. S. (1996). Complete PCB congener distributions for 17Aroclor mixtures determined by 3 HRGC systems optimized for comprehensive, quantitative, congener-specific analysis. J. High Resolut. Chromatogr. 19, 657– 668. Gospe, Jr., S. M., and Calaban, M. J. (1988). Central nervous system distribution of inhaled toluene. Fundam. Appl. Toxicol. 11, 540 –545. Hansen, L. G. (1987). Food chain modification of the composition and toxicity of polychlorinated biphenyl (PCB) residues. Rev. Environ. Toxicol. 3, 149 – 212. Hansen, L. G. (1998). Stepping backward to improve assessment of PCB congener toxicities. Environ. Health Perspect. 106(Suppl. 1), 171–189. Kafafi, S. A., Afeefy, H. Y., Ali, A. H., Said, H. K., and Kafafi, A. G. (1993). Binding of polychlorinated biphenyls to the aryl hydrocarbon receptor. Environ. Health Perspect. 101, 422– 428. Kodavanti, P. R. S., 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
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changes in intracellular Ca21 buffering and protein kinase C activity in the absence of changes in tyrosine hydroxylase. Toxicol. Appl. Pharmacol. 153, 186 –198. Kodavanti, P. R. S., Shafer, T. J., Ward, T. R., Mundy, W. R., Freudenrich, T., Harry, G. J., and Tilson, H. A. (1994). Differential effects of PCB congeners on phosphoinositide hydrolysis and protein kinase C translocation in rat cerebellar granule cells. Brain Res. 662, 75– 82. Kodavanti, P. R. S., Shin, D., Tilson, H. A., and Harry, G. J. (1993). Comparative effects of two polychlorinated biphenyl congeners on calcium homeostasis in rat cerebellar granule cells. Toxicol. Appl. Pharmacol. 123, 97–106. Kodavanti, P. R. S., and Tilson, H. A. (1997). Structure–activity relationships of potentially neurotoxic PCB congeners in the rat. Neurotoxicology 18, 423– 438. Kodavanti, P. R. S., Ward, T. R., McKinney, J. D., and Tilson, H. A. (1995). Increased [3H]phorbol ester binding in rat cerebellar granule cells by PCB mixtures and congeners: Structure–activity relationships. Toxicol. Appl. Pharmacol. 130, 140 –148. Kodavanti, P. R. S., Ward, T. R., McKinney, J. D., and Tilson, H. A. (1996). Inhibition of microsomal and mitochondrial Ca21-sequestration in rat cerebellum by polychlorinated biphenyl mixtures and congeners: Structure– activity relationships. Arch. Toxicol. 70, 150 –157. Koss, G., Meyer-Rogge, D., Seubert, S., Seubert, A., and Losekam, M. (1993). 2,29,39,4,49,5,59-heptachlorobiphenyl (PCB 180)— on its toxicokinetics, biotransformation and porphyrinogenic action in female rats. Arch. Toxicol. 67, 651– 654. Kuratsune, M., and Shapiro, R. E. (1984). PCB poisoning in Japan and Taiwan. Prog. Clin. Biol. Res. 137, 1–155. Kuwabara, K., Yakushiji, T., Watanabe, I., Yoshida, S., Koyama, K., Kunita, N., and Hara, I. (1978). Relationship between breast feeding and PCB residues in blood of the children whose mothers were occupationally exposed to PCBs. Int. Arch. Occup. Environ. Health 41, 189 –197. Masuda, Y., Kagawa, H., Tokudome, S., and Kuratsune, M. (1979). Transfer of various polychlorinated biphenyls to the fetuses and offspring of mice. Food Cosmet. Toxicol. 17, 623– 627. Masuda, Y., Kuroki, H., Yamaryo, T., Haraguchi, K., Kuratsune, M., and Hsu, S. (1982). Comparison of causal agents in Taiwan and Fukuoka PCB poisonings. Chemosphere 11, 199 –206. Mattson, M. P. (1991). Evidence for the involvement of protein kinase C in neurodegenerative changes in cultured human cortical neurons. Exp. Neurol. 112, 95–103. McKinney, J, D., and Waller, C. L. (1994). Polychlorinated biphenyls as hormonally active structural analogs. Environ. Health Perspect. 102, 290 – 297. Ness, D. K., Schantz, S. L., and Hansen, L. G. (1994). PCB congeners in the rat brain: Selective accumulation and lack of regionalization. J. Toxicol. Environ. Health 43, 453– 468. Patterson, Jr., D. G., Todd, G. D., Turner, W. E., Maggio, V., Alexander, L. R., and Needham, L. L. (1994). Levels of non-ortho-substituted (coplanar), mono- and di-ortho-substituted polychlorinated biphenyls, dibenzo-p-dioxins, and dibenzofurans in human serum and adipose tissue. Environ. Health Perspect. 102, 195–204. Safe, S. H. (1994). Polychlorinated biphenyls (PCBs): Environmental impact,
biochemical and toxic responses, and implications for risk assessment. Crit. Rev. Toxicol. 24, 87–149. Schantz, S. L., Moshtaghian, J., and Ness, D. K. (1995). Spatial learning deficits in adult rats exposed to ortho-substituted PCB congeners during gestation and lactation. Fundam. Appl. Toxicol. 26, 117–126. Schecter, A., Stanley, J., Boggess, K., Masuda, Y., Mes, J., Wolff, M., Furst, P., Furst, C., Wilson-Yang, K., and Chisholm, B. (1994). Polychlorinated biphenyl levels in the tissues of exposed and non-exposed humans. Environ. Health Perspect. 102, 149 –158. Schultz, D. E., Petrick, G., and Duinker, J. C. (1989). Complete characterization of polychlorinated biphenyl congeners in commercial Aroclor and Clophen mixtures by multidimentional gas chromatography-electron capture detection. Environ. Sci. Technol. 23, 852– 859. Seegal, R. F., Bush, B., and Brosch, K. O. (1991). Sub-chronic exposure of the adult rat to Aroclor 1254 yields regionally-specific changes in central dopaminergic function. Neurotoxicology 12, 55– 66. Seegal, R. F., and Schantz, S. L. (1994). Neurochemical and behavioral sequelae of exposure to dioxins and PCBs. In Dioxins and Health (A. Schecter, Ed.), pp. 409 – 447. Plenum Press, New York. Seegal, R. F., and Shain, W. (1992). Neurotoxicity of polychlorinated biphenyls: The role of ortho-substituted congeners in altering neurochemical function. In The Vulnerable Brain and Environmental Risks, Vol. 2. Toxins in Food (R. L. Isaacson and K. F. Jensen, Eds.), pp. 169 –195. Plenum Press, New York. Shafer, T. J., Mundy, W. R., Tilson, H. A., and Kodavanti, P. R. S. (1996). Disruption of inositol phosphate accumulation in cerebellar granule cells by polychlorinated biphenyls: A consequence of altered Ca21 homeostasis. Toxicol. Appl. Pharmacol. 141, 448 – 455. Shain, W., Overmann, S. R., Wilson, L. R., Kostas, J., and Bush, B. (1986). A congener analysis of polychlorinated biphenyls accumulating in rat pups after perinatal exposure. Arch. Environ. Contam. Toxicol. 15, 687–707. Smith, A., Schloemer, J., Lowry, L., Smallwood, A., Ligo, R., Tanaka, S., Stringer, W., Jones, M., Hervin, R., Glueck, C. (1982). Metabolic and health consequences of occupational exposure to polychlorinated biphenyls. Br. J. Ind. Med. 39, 361–369. Tanabe, S., Nakagawa, Y., and Tatsukawa, R. (1981). Absorption efficiency and biological half-life of individual chlorobiphenyls in rats treated with Kanechlor products. Agric. Biol. Chem. 45, 717–726. Tilson, H. A., and Kodavanti, P. R. S. (1997). Neurochemical effects of polychlorinated biphenyls: An overview and identification of research needs. Neurotoxicology 18, 727–744. U.S. EPA (United States Environmental Protection Agency) (1993). Workshop Report on Developmental Neurotoxic Effects Associated with Exposure to PCBs. Prepared by Eastern Research Group, Inc., Lexington, MA. Prepared for Risk Assessment Forum, EPA/630/R-92/004. World Health Organization (1993). Environmental Health Criteria 140: Polychlorinated Biphenyls and Terphenyls, 2nd ed. Prepared by S. Dobson and G.J. van Esch. International Programme on Chemical Safety, Geneva, Switzerland. Wyss, P. A., Muhlebach, S., and Bickel, M. M. (1986). Long-term pharmacokinetics of 2,29,4,49,5,59-hexachlorobiphenyl (6-CB) in rats with constant adipose tissue mass. Drug Metab. Dispos. 14, 361–365.