Repeated Exposure of Adult Rats to Aroclor 1254 Causes Brain Region-Specific Changes in Intracellular Ca2+Buffering and Protein Kinase C Activity in the Absence of Changes in Tyrosine Hydroxylase

TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.

153, 186 –198 (1998)

TO988533

Repeated Exposure of Adult Rats to Aroclor 1254 Causes Brain Region-Specific Changes in Intracellular Ca21 Buffering and Protein Kinase C Activity in the Absence of Changes in Tyrosine Hydroxylase1,2 Prasada Rao S. Kodavanti, Ethel C. Derr-Yellin, William R. Mundy, Timothy J. Shafer, David W. Herr, Stanley Barone, Jr., Neepa Y. Choksi, Robert C. MacPhail, and Hugh A. Tilson Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711 Received December 3, 1997; accepted July 17, 1998

Repeated Exposure of Adult Rats to Aroclor 1254 Causes Brain Region-Specific Changes in Intracellular Ca21 Buffering and Protein Kinase C Activity in the Absence of Changes in Tyrosine Hydroxylase. Kodavanti, P. R. S., Derr-Yellin, E. C., Mundy, W. R., Shafer, T. J., Herr, D. W., Barone, S., Jr., Choksi, N. Y., MacPhail, R. C., and Tilson, H. A. (1998). Toxicol. Appl. Pharmacol. 153, 186 –198. Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants, some of which may be neurotoxic. In vitro studies from this laboratory indicated that noncoplanar PCBs perturbed intracellular signal transduction mechanisms including Ca21 homeostasis, receptor-mediated inositol phosphate production, and translocation of protein kinase C (PKC). In the present study, we examined the effects of PCBs in vivo by dosing adult male Long– Evans rats orally with Aroclor 1254 (0, 10, or 30 mg/kg/day; 5 days/week for 4 weeks) in corn oil. At 24 h after the last dose, rats were tested for motor activity in a photocell device for 30 min. Immediately, the rats were euthanized, blood was collected for thyroid hormone analysis, and brains were removed, dissected into regions (cerebellum, frontal cortex, and striatum), and subcellular fractions were obtained for neurochemical analysis. Following Aroclor 1254 treatment, body weight gain in the high-dose group was significantly lower than the control and low-dose groups. Horizontal motor activity was significantly lower in rats dosed with 30 mg/kg Aroclor 1254. Ca21 buffering by microsomes was significantly lower in all three brain regions from the 30 mg/kg group. In the same dose group, mitochondrial Ca21 buffering was affected in cerebellum but not in cortex or striatum. Similarly, total cerebellar PKC activity was decreased significantly while membrane-bound PKC activity was significantly elevated at 10 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. 0041-008X/98

and 30 mg/kg. PKC activity was not altered either in cortex or the striatum. Neurotransmitter levels in striatum or cortex were slightly altered in PCB-exposed rats compared to controls. Furthermore, repeated oral administration of Aroclor 1254 to rats did not significantly alter forebrain tyrosine hydroxylase immunoreactivity or enzymatic activity. Circulating T4 (total and free) concentrations were severely depressed at both doses in Aroclor 1254-exposed rats compared to control rats, suggesting a severe hypothyroid state. These results indicate that (1) in vivo exposure to a PCB mixture can produce changes in second messenger systems that are similar to those observed after in vitro exposure of neuronal cell cultures; (2) second messenger systems seem to be more sensitive than alterations in neurotransmitter levels or tyrosine hydroxylase involved in dopamine synthesis during repeated exposure to PCBs; and (3) the observed motor activity changes were independent of changes in striatal dopamine levels.

Polychlorinated biphenyls (PCBs) belong to a large group of persistent chemicals known as halogenated aromatic hydrocarbons, which also include other environmentally relevant compounds such as polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and polychlorinated diphenyl ethers (Erickson, 1986). Because of their lipophilic nature, these compounds bioaccumulate and have been detected in fish, wildlife, and various human body fluids and tissues (Safe et al., 1987; WHO, 1993). There is considerable concern over the potential adverse health effects associated with exposure to these chemicals. For example, there have been two major accidental PCB poisoning incidents (“Yusho” in Japan and “YuCheng” in Taiwan) where more than 1000 people had chloracne, numbness, weakness in limbs, and/or decreased peripheral nerve conduction velocities (Rogan et al., 1988). Acute and long-term exposure to PCBs has been reported to cause neurological and nonspecific psychological or psychosomatic effects, such as headache, dizziness, nausea, depression, sleep and memory disturbances, nervousness, fatigue, and

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impotence (Chia and Chu, 1984; WHO/EURO, 1987; Schantz, 1996). Fischbein et al. (1979) and Seppalainen et al. (1985) have also reported these symptoms in capacitor-manufacturing workers exposed to PCBs for extended periods. There are also reports indicating that PCB exposure may produce developmental neurotoxicity. Children exposed in utero showed subtle developmental delays, altered neurological function, and cognitive deficits (Jacobson et al., 1985; Rogan and Gladen, 1992; Huisman et al., 1995; Jacobson and Jacobson, 1996). In laboratory animals, developmental exposure to PCBs is reported to alter cognitive and motor functions (Tilson et al., 1979; Schantz et al., 1989, 1991, 1995). The cellular mechanism(s) by which PCBs affect the nervous system in adults as well as developing animals is not known. Seegal and his colleagues (1985, 1986a,b, 1991a,b) have shown alterations in rodent brain dopamine levels following exposure to commercial PCB mixtures. Ortho-substituted PCB congeners accumulate in the brain and decrease brain dopamine levels in adults (Shain et al., 1986) and developing animals (Seegal, 1996). Shain et al. (1991) reported that orthosubstituted PCBs decrease dopamine levels in PC12 cells in vitro and suggested that decreased brain dopamine levels in vivo might be due to ortho PCB congeners. Other studies indicate that PCBs may also affect thyroid hormone function. For example, Byrne et al. (1987), Ness et al. (1993), and Morse et al. (1993, 1996) reported that PCBs decrease circulating thyroid hormone levels dramatically. Altered thyroid hormone status during development is known to produce motor incoordination, severe hearing loss, and memory deficits (Porterfield, 1994). Recently, Goldey et al. (1995) reported that ototoxic effects of PCBs are associated with decreased circulating thyroid hormones following perinatal exposure. Schantz et al. (1995) reported that rats exposed from gestational day (GD)10 to GD16 to 2,4,49-trichlorobiphenyl, 2,39,4,49,5-pentachlorobiphenyl or 2,29,4,49,5,59-hexachlorobiphenyl showed deficits on a T-maze delayed spatial alternation task in adulthood. However, decreases in thyroid hormone levels were observed only with the latter two congeners, suggesting that PCBs may influence learning and memory through mechanisms other than alterations in thyroid hormone function. To characterize the cellular actions of PCBs more completely, our laboratory has explored the effects of PCBs on signal transduction processes, including Ca21 homeostasis and inositol phosphate turnover. We reported that 2,29-dichlorobiphenyl (2,29-DCB), a noncoplanar congener, increased intracellular free Ca21 ([Ca21]i), inhibited 45Ca21 uptake by mitochondria and microsomes, altered receptor-mediated inositol phosphate production, and induced protein kinase C (PKC) translocation at low micromolar concentrations in primary cultures of cerebellar granule cells (Kodavanti et al., 1993, 1994). 2,29-DCB was cytotoxic at higher concentrations (.100 mM). Comparatively, the coplanar congener 3,39,4,49,5-pentachlorobiphenyl did not alter signal transduction processes and was not cytotoxic (Kodavanti et al., 1993, 1994). Further

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structure–activity relationship (SAR) studies indicated that the in vitro activity of PCB congeners was associated with chlorine substitution patterns favoring noncoplanarity (Kodavanti et al., 1995, 1996a,b). These in vitro studies suggest that signal transduction processes, involved in the normal function of neurons, are sensitive targets for noncoplanar PCBs. We have extended these in vitro studies by dosing adult rats repeatedly with a commercial PCB mixture, Aroclor 1254. The objectives of the present study were (1) to determine whether changes in Ca21 homeostasis and PKC distribution observed after in vitro exposure also occur after exposure in vivo in adult animals and (2) to examine the effect of repeated exposure to PCBs on other proposed mechanisms of PCB-induced neurotoxicity such as brain catecholamines and circulating thyroid hormones in adult rats. MATERIALS AND METHODS Chemicals. A commercial PCB mixture, Aroclor 1254, (purity . 99%; lot no. 6024) was purchased from AccuStandard Inc. (New Haven, CT). Radiolabeled 45Ca21 (34.12 mCi/mg; purity . 99%) as CaCl2, 14C-L-tyrosine (54.8 mCi/mmol; purity . 97%), and 32P-ATP (10 Ci/mmol; purity . 99.9%) were purchased from Dupont New England Nuclear Corporation (Boston, MA). Radioimmunoassay kits for thyroid hormone measurements were purchased from Diagnostic Products Inc. (Los Angeles, CA). All the 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. Environmental Protection Agency. 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 (7:00 –19:00 h). In vivo dosing. After 1 week of acclimation, rats were dosed orally by gavage with Aroclor 1254 in corn oil (2 ml/kg). The congener-specific analysis of this mixture is given in the companion paper (Kodavanti et al., 1998). The selected dosages were 0, 10, or 30 mg/kg/day based on previous experiments where no mortality was observed at these doses (Nishida et al., 1997). The number of rats dosed are 26, 25, and 35 for 0, 10, or 30 mg/kg Aroclor 1254, respectively. The rats were dosed once a day, 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. At 24 h after the last dosage, all the rats were tested behaviorally and immediately euthanized by decapitation. Blood was collected for thyroid hormone analysis, and the brains were removed and dissected into different brain regions by a technique slightly modified from that of Glowinski and Iversen (1966). The striatum and the cortex consisted of one hemisphere of cortical tissue, posterior to the cut at the level of the optic chiasm, after the removal of hippocampus were quick frozen on dry ice, and stored at 280°C until neurotransmitter analysis, usually within 2 weeks. Frontal cortex, cerebellum, and striatum were fractionated immediately for Ca21 buffering and PKC. For immunohistochemistry, six control and six treated (30 mg/kg dose group) rats were anesthetized with sodium pentobarbital (100 mg/kg ip) and perfused intracardially with ice-cold saline followed by phosphate-buffered 4% paraformaldehyde. Brains were removed, postfixed for 2 h in the same solution, and then transferred into a solution of 30% sucrose in 0.1 M phosphate-buffered saline (PBS, pH 5 7.4) before sectioning (40 mm) on a freezing stage microtome. Motor activity. Horizontal and vertical motor activity were measured during a 30-min session (MacPhail et al., 1989) in six automated motor activity

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devices (Motron Motility Meter, Stockholm, Sweden). Each device was comprised of a 20.5 3 32 cm translucent platform that housed a 5 3 8 matrix of photodetectors. Illumination in each of the devices was provided by a single overhead 30-W incandescent lamp. Horizontal movements that occluded a detector were recorded as horizontal activity. In addition, an array of six photoemitters and detectors was placed 16 cm above the platform and used to record vertical activity. Animals were placed in a Plexiglas chamber (22 3 33.5 3 29 cm) positioned over each platform. Each device was housed in a larger ventilated sound- and light-attenuating enclosure. Programming and data-recording equipment were located in an adjacent room. After each test session, droppings were removed and urine pools were wiped clean prior to testing the next rat. Isolation of mitochondria and microsomes. Fractionation was performed according to the procedures of Gray and Whittaker (1962) and Dodd et al. (1981) with slight modifications (Kodavanti et al., 1993). Brain regions (frontal cortex, cerebellum, or striatum) were homogenized in 9 vol of cold buffer containing 250 mM sucrose, 5 mM Hepes, 10 mM KCl, and 1 mM MgCl2, pH 7.05. The homogenate was centrifuged at 1000g for 10 min, the pellet was discarded, and the resulting supernatant was centrifuged at 9000g for 20 min. The supernatant was then centrifuged at 105,000g for 60 min and the microsomal pellet was resuspended in homogenizing buffer. The P2 pellet was suspended in 10 ml of 0.32 M sucrose, layered over 1.2 M sucrose (10 ml), and centrifuged at 150,000g for 20 min (Beckman model L8 –70, rotor Ti 50.2). The resulting mitochondrial pellet was resuspended in homogenizing buffer. Protein content was determined by the method of Lowry et al. (1951). Freshly isolated microsomes and mitochondria were assayed for 45Ca21 uptake. 45 Ca21 uptake by mitochondria and microsomes. Uptake of 45Ca21 was measured as outlined by Moore et al. (1975). The assay mixture of 1.5 ml contained 30 mM histidine-imidazole buffer (pH 6.8), 100 mM KCl, 5 mM MgCl2, 5 mM sodium azide (added only for microsomal calcium uptake), 5 mM ammonium oxalate, mitochondrial (180 mg) or microsomal (50 mg) protein, and 0.1 mCi of 45CaCl2 containing 5 mM free Ca21 in calcium– ethylene glycol-bis(b-aminoethyl ether)N,N,N9,N9-tetraacetic acid (Ca21– EGTA)-buffered medium. The concentrations of free Ca21 were calculated according to Fabiato and Fabiato (1978). The tubes were preincubated at 37°C for 5 min. Uptake of 45Ca21 was initiated by addition of ATP (5 mM final concentration) and continued for 20 min at 37°C. After the 20-min incubation, the samples were filtered through a 0.45-mm Millipore filter. After washing the filters twice with 5 ml of 10 mM Tris (pH 7.4), they were transferred directly to vials containing 10 ml of Ultima Gold scintillation fluid and the amount of radioactivity was determined by liquid scintillation spectroscopy. Nonspecific 45 Ca21 binding was studied in the absence of ATP and subtracted from total uptake to get the specific uptake of 45Ca21, expressed as pmol/mg protein/min.

Protein kinase C assay. Cytosolic and membrane fractions of brain tissue homogenates were prepared according to the method of Malkinson et al. (1984). Brain tissues (cerebellum, frontal cortex, or striatum) were homogenized (100 mg/ml) in ice-cold homogenizing buffer containing 20 mM Tris– HCl, 2 mM EDTA, 2 mM EGTA, 50 mM b-mercaptoethanol, 0.5 mM phenylmethylsulfonyl-fluoride, 0.02 mM leupeptin, and 320 mM sucrose, pH 7.5. The homogenate was centrifuged for 30 min at 100,000g in a Ti50 rotor with a Beckman L8 –70 ultracentrifuge. The supernatant was held in reserve, the pellet was resuspended in homogenization buffer (100 mg/ml), and the centrifugation step was repeated. The supernatants were combined to make up the cytosolic fraction and the pellet was resuspended in 1 ml of homogenization buffer containing 0.2% Triton X-100, sonicated, and incubated on ice for 30 min. The homogenate was then centrifuged at 100,000g for 30 min and the resulting supernatant was saved as membrane fraction. The protein content was determined by the Bradford (1976) assay. PKC activity was determined by measuring the incorporation of 32P from g-[32P]ATP into the PKC-specific substrate, neurogranin (Chen et al., 1993). Neurogranin28 – 43 (NG28 – 43) was developed as a selective and potent peptide for use as an exogenous substrate for in vitro assays of PKC activity. It is selective for PKC and can be used in crude tissue homogenates (Gonzalez et al., 1993). To measure PKC activity, 10 mg of soluble or membrane protein

was added to solution containing 20 mM Tris–HCl, 5 mM MgCl2, 10 mM NG, 0.5 mM CaCl2, 40 mg/ml dioctanoylglycerol, and 40 mg/ml phosphatidylserine, pH 7.5. Following 5 min preincubation in a 35°C water bath, the assay was initiated by the addition of 5 nmol/sample of ATP containing 0.5 mCi g-[32P]ATP. The final volume of each sample was 250 ml. After 5 min, the assay was terminated by adding 1 ml of ice-cold 25% trichloroacetic acid and placing samples on ice. Each sample was filtered onto a 0.45-mm membrane filter and the sample tube was rinsed with 1 ml of trichloroacetic acid solution. Filters were then washed twice with 2 ml of trichloroacetic acid and placed into a 7-ml scintillation vial. Five milliliters of Ultima Gold scintillant were added and samples were counted in a Beckman scintillation counter. Values from triplicate measurements were averaged for calculation of PKC activity, expressed as nmol/min/mg protein. Nonspecific kinase activity, measured in the absence of Ca21 and phospholipids, was always less than 5% of total activity. Analysis of neurotransmitters. High-pressure liquid chromatography was used to quantify norepinephrine (NE), dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and serotonin (5-HT) and its metabolite 5-hydroxyindolacetic acid (5-HIAA) in both the cortex and striatum. Each sample was sonicated on ice for 5 to 10 s at 53 W in 1 ml of 0.1 N perchloric acid containing 10 mM EDTA and the internal standard N-methyl-5-hydroxytryptamine (NM-5-HT). The concentration of NM-5-HT was always kept constant: 2.6283 mM for the striatum and 1.3141 mM for the cortex. The samples were then centrifuged at 4°C for 10 min (12,405g) and 50 ml of the supernatant was injected into the HPLC system. The HPLC system consisted of a Waters 510 pump, a Waters 712 WISP injector (cooled to 4°C), a Waters 10-mm Bondpack C18 precolumn (Waters Corp., Milford, MA), and a Rainin 5-mm Microsorb C18 column (4.6 3 250 mm) (Rainin Instrument Co., Woburn, MA), which was thermostated to 40°C. Electrochemical detection was performed with an ESA 5100A detector, a guard cell (Model 5020) set at 10.45V, and a Model 5011 detector with detectors 1 and 2 set at 10.05 and 10.42V, respectively (ESA Inc., Chelmsford, MA). The mobile phase contained 80 mM H3PO4 (Fisher Scientific, Fair Lawn, NJ), 2.5 mM heptanesulfonic acid (Sigma Chemical Co., St. Louis, MO), 10 mM EDTA (Sigma), and 12% methanol (Fisher), with an apparent pH of 2.8 and a flow rate of 1.2 ml/min. Data collection and analysis were performed with a Waters Maxima 820 system using area ratios to determine sample concentrations. Standards for NE, DA, DOPAC, HVA, 5-HT, 5-HIAA, and NM-5-HT were purchased from Sigma Chemical Company. The perchloric acid-extracted tissue pellet was suspended and digested in 0.1 N NaOH, diluted with water to 0.01 N NaOH, and analyzed for protein content by the BCA protein assay (Pierce, Rockford, IL) in a Thermomax microplate reader with SOFTmax software (Molecular Devices, Sunnyvale, CA). Bovine serum albumin (RIA grade, Sigma) was used as the protein standard. Data are expressed as ng/mg protein 6 SE. Tyrosine hydroxylase activity in striatal minces. Tyrosine hydroxylase activity, the rate-limiting step in biosynthesis of catecholamines, was measured in minces of rat corpus striatum as an assessment of dopamine synthesis. A radiometric assay was used to measure formation of 14CO2 evolved during the decarboxylation of L-dihydroxyphenylalanine (DOPA) to form dopamine, starting from [1-14C]-L-tyrosine, as described in detail previously (Waymire et al., 1971; Booth et al., 1990). Briefly, corpus striatum was minced to produce a fine slurry and then was suspended (10 mg/ml) in modified Kreb’s solution containing 124 mM NaCl, 0.5 mM KCl, 0.8 mM CaCl2 z 2H20, 1.3 mM MgCl2 z 6H20, 1.4 mM KH2PO4, 80 mM sucrose, and 20 mM HEPES aerated with O2/CO2 (95/5) to pH 7.0. Aliquots (300 ml) of the tissue suspension were added to tubes containing test agents and the reaction was initiated by addition of 100 ml [1-14C]-L-tyrosine (0.1 mCi). The reaction mixture was capped with a vial containing a phenethylamine-saturated filter strip (0.5 3 6 cm) to trap evolved 14CO2 and then was incubated for 30 min at 37°C. Following incubation, the reaction was terminated by addition of 1 N perchloric acid (1 ml). Adsorbed 14CO2 on the filter strips was quantitated by liquid scintillation spectroscopy (14C counting efficiency 85%). Appropriate blanks (tissue omitted) were maintained and subtracted from sample values to determine basal (control) tyrosine hydroxylase activity, which averaged 4.9 6 0.4 pmol

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CO2/mg protein/min. Protein concentrations were determined by the method of Lowry et al. (1951). Forskolin (1 mM), an activator of tyrosine hydroxylase activity, stimulated enzyme activity to 50% above basal level (Onali and Olianas, 1987). Comparatively, a-methyl tyrosine (25 mM), a competitive inhibitor of tyrosine hydroxylase, inhibited enzyme activity to 75% below basal level (Nagatsu et al., 1964). Immunohistochemistry. Immunohistochemistry was performed with a polyclonal antiserum to tyrosine hydroxylase (Eugene Tech, NJ) diluted 1:10,000 followed by immunoperoxidase techniques on 40-mm free-floating coronal sections from perfusion-fixed brains. Sections were incubated for 30 min at room temperature on a shaker in 10% Normal Goat Serum (NGS) with 0.5% Triton X-100 in PBS (PBS-T). Sections were then transferred to polystyrene sample tubes, with polyclonal antiserum with 3% NGS in PBS-T, and exposed to ultrasonic irradiation in an ultrasonic bath (Crest Ultrasonics, Trenton, NJ) of cold water (15°C) for 30 s, then incubated for 18 h at 4°C on a shaker in a humid environment. After several washes, the bound antibodies were labeled with a biotinylated goat anti rabbit serum for 1 h, washed, and incubated with an avidin– biotin peroxidase solution (Elite Vectastain, Vector, Burlingame, CA) for 1 h. The immunoperoxidase label was visualized with 0.5% 3,39 diaminobenzidine tetrahydrochloride (Polyscience, Warrington, PA) with 0.003% H2O2 and intensified with nickel ammonium sulfate and cobalt chloride. The sections were then rinsed thoroughly and mounted on gelatincoated slides. The sections were dried overnight, dehydrated through a graded series of alcohols, cleared in xylene, and cover slipped. Specificity of the immunohistochemical reaction was confirmed by the absence of labeled profiles in tissue sections where the primary or secondary antisera were omitted (data not shown). Immunohistochemically stained sections were examined qualitatively in a rostral to caudal gradient by an observer blind to the treatment condition of each animal. Circulating thyroid hormones. Total T4, free T4, total T3, and free T3 were determined with the standard radioimmunoassay kits (Diagnostic Products Corp.) based on the competitive protein binding technique. Blood was collected and allowed to clot on ice for 1 h. The blood samples were centrifuged at 750 g for 15 min to separate serum. Serum samples were stored at 280°C until radioimmunoassay. Statistics. The neurotransmitter data were analyzed in each brain region by a multivariate analysis of variance (MANOVA) with Pillai’s Trace as the F statistic (Olson, 1976, 1979; Pillai, 1955). To control the Type I error rate, the critical a for each brain region was set to a 5 0.025 (0.05/two regions). To assess regional differences for each neurotransmitter/metabolite, a repeated measures analysis of variance (ANOVA) was performed with a critical a 5 0.0083 (0.05/six compounds). Degrees of freedom for within subject factors and their interactions were adjusted with a Greenhouse Geisser correction factor (Geisser and Greenhouse, 1958; Greenhouse and Geisser, 1959; Keselman and Rogan, 1980). Significant overall effects were further analyzed by step-down ANOVAs, which used Bonferroni corrections to maintain a familywise a # 0.05. Significant differences between treatment groups were determined with a Tukey-Kramer multiple comparison test (a 5 0.05) (Kramer, 1956). The stringent a correction procedures employed prevented an excessive Type I error rate. However, such procedures may result in decreased statistical power, thus increasing the Type II error rate (Muller et al., 1983). Therefore, if an overall effect was significant, and subsequent step-down ANOVAs failed to reach the corrected significance level (but had an a # 0.05), the actual probability values are reported. This allows readers to apply their own judgement as to the biological significance of the results. Body weight gain data were analyzed by a two-way ANOVA with day (Days 1–25) as one factor and dose (0, 10, or 30 mg/kg) as the other, followed by Dunnett’s post-hoc test. All analyses were performed with PROC GLM in SAS (SAS Institute Inc., 1989). All other data were analyzed by a one-way ANOVA followed by Tukey’s multiple comparison test. The accepted level of significance was p , 0.05.

FIG. 1. Changes in body weight of rats exposed to Aroclor 1254 (Ar; dose levels: 0, 10, or 30 mg/kg/day) for 4 weeks (5 days/week). Values are means 6 SEM of 25, 26, and 35 rats for 0, 10, and 30 mg/kg doses, respectively. * Significantly different from control at p , 0.05.

RESULTS

Effects on Body Weight Gain and Motor Activity The ANOVA of body weight gain data indicated a significant effect of day (F[7,581] 5 408, p , 0.0001), dose (F[2,83] 5 28.1, p , 0.0001), and an interaction between day and dose (F[14,581] 5 63.7, p , 0.0001). Body weight gain in both treated groups was not altered until after 1 week of exposure. In the 10 mg/kg dose group, body weight gain was slightly lower than controls after 3 weeks of exposure (Fig. 1). Body weight gain of rats from the high-dose group was significantly lower than controls and low-dose groups after 8 to 12 days of exposure. Rats from the high-dose group did not continue to gain weight after 1 week of exposure to Aroclor 1254 (Fig. 1) Therefore, we conducted a weight gain-control experiment and the results are given at the end of Results. The ANOVA on motor activity data indicated a significant effect of treatment on horizontal activity (F[2,69] 5 13.37, p , 0.0001), but not on vertical activity (Fig. 2). Horizontal motor activity was significantly lower in rats exposed to 30 mg/kg Aroclor 1254 (Fig. 2) compared to control and 10 mg/kg Aroclor 1254-dosed rats. These results indicate that repeated exposure of Aroclor 1254 at 30 mg/kg dose causes decreased weight gain and hypoactivity in rats. These results are in agreement with previous reports in rats (Nishida et al., 1997) and mice (Rosin and Martin, 1981). Effects on Ca21 Buffering and PKC Previous in vitro studies indicated that intracellular Ca21 buffering and PKC are affected by micromolar concentrations of noncoplanar PCBs (Kodavanti et al., 1993, 1994). In the present in vivo study, intracellular Ca21 buffering was deter-

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mined by measuring the uptake of 45Ca21 by microsomes and mitochondria. The ANOVA on microsomal 45Ca21 uptake data indicated a significant effect of treatment in cerebellum (F[2,9] 5 4.67, p , 0.04), frontal cortex (F[2,9] 5 11.1, p , 0.004), and striatum F[2,9] 5 5.32, p , 0.03). 45Ca21 uptake by microsomes was significantly lower in all three brain regions of the 30 mg/kg group (Fig. 3) compared to controls. In the 30 mg/kg dose group, mitochondrial Ca21 buffering was affected only in cerebellum (F[2,9] 5 11.99, p , 0.003) (Fig. 3). The ANOVA on PKC activity data indicated a significant effect of treatment only in cerebellum. In cerebellum, total PKC activity was decreased significantly (F[2,9] 5 17.7, p , 0.0008), while membrane-bound PKC activity was elevated (F[2,9] 5 22.9, p , 0.0003) in 10 and 30 mg/kg dose groups compared to control group (Fig. 4). Total or membrane-bound PKC activity was not altered in cortex or striatum in any dose groups (Fig. 4). These results indicate that in vivo exposure to a PCB mixture can produce changes in intracellular Ca21 buffering and PKC activity similar to those observed after in vitro exposure of neuronal cell cultures to individual as well as mixtures of PCB congeners. Effects on Neurotransmitter Concentrations FIG. 2. Changes in motor activity (horizontal and vertical counts) of rats following exposure to Aroclor 1254 (0, 10, or 30 mg/kg/day) for 4 weeks (5 days/week). Values are means 6 SEM of 24 rats. *Significantly different from control at p , 0.05.

The cortex and striatum were chosen for determination of catecholamine levels due to previously reported changes in striatal dopaminergic function following PCB exposure

FIG. 3. Intracellular calcium buffering in cerebellum, frontal cortex, and striatum of rats following exposure to Aroclor 1254 (0, 10, or 30 mg/kg/day) for 4 weeks (5 days/week). Microsomes or mitochondria, isolated from different brain regions, were incubated in 30 mM histidine–imidazole buffer with 0.1 mCi 45 CaCl2 (5 mM). After incubation, the samples were filtered rapidly through a 0.45-mm millipore filter and the amount of radioactivity on the filters was determined by liquid scintillation spectroscopy. Values are means 6 SEM of four rats. Each fraction obtained from each rat was assayed in triplicate samples. * Significantly different from control at p , 0.05. For striatal 45Ca21-uptake studies, tissue from three animals was pooled in each experiment.

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FIG. 4. PKC activity in cerebellum, frontal cortex, and striatum of rats following exposure to Aroclor 1254 (0, 10, or 30 mg/kg/day) for 4 weeks (5 days/week). Values are means 6 SEM of four rats. Each fraction obtained from each rat was assayed in triplicate samples. *Significantly different from control at p , 0.05.

(Seegal et al., 1986a, 1991a) and to allow for assessment of regional specificity of neurochemical changes produced by repeated PCB exposure (Seegal et al., 1985, 1986b). Treatment with Aroclor 1254 had no effect on any of the neurotransmitters, but caused minor increases in several of the metabolites that were assayed (Table 1). There were no treatment-related effects on regional brain weight (or percent protein content), which averaged 209.8 6 6.3 mg (7.8 6 0.2%) and 66.1 6 5.6

mg (8.8 6 0.2%) for the cortex and striatum, respectively. The MANOVA did not indicate any treatment-related change in any neurotransmitter/metabolite in the cortex (F[14,16] 5 1.2311, p 5 0.3419). Additionally, in cortical tissue there were no changes in any individual substances due to treatment with Aroclor 1254 (F’s[2,13] # 3.38, p’s $ 0.0657). The MANOVA for an overall treatment effect in the striatum (F[14,16] 5 2.5285, p 5 0.0390) failed to reach corrected

TABLE 1 Neurotransmitter and Metabolite Concentrations in Cortex and Striatum of Rats Repeatedly Exposed to Aroclor 1254 Neurotransmittera Region Cortex

Striatum

Doseb

NEc

DAc

DOPACc

HVAc

5-HT

5-HIAAc

0 (6) 10 (4) 30 (6) 0 (6) 10 (4) 30 (6)

5.03 6 0.37 6.27 6 0.47 5.81 6 0.37 1.50 6 0.22 2.71 6 1.34 2.01 6 0.32

4.01 6 0.79 6.16 6 0.94 5.19 6 1.07 105.04 6 6.36 114.21 6 2.44 115.34 6 3.20

0.93 6 0.13 1.26 6 0.06 1.10 6 0.15 22.18 6 1.63 32.88 6 2.28* 28.83 6 1.71*

0.51 6 0.07 0.74 6 0.08 0.62 6 0.11 11.70 6 0.90 12.85 6 0.32 12.53 6 0.95

5.19 6 0.39 5.67 6 0.31 5.52 6 0.26 5.56 6 0.38 5.43 6 0.78 5.72 6 0.30

3.75 6 0.27 4.32 6 0.21 4.41 6 0.24 6.77 6 0.37 8.16 6 0.40 8.93 6 0.43*

Data are mean (ng/mg protein) 6 SEM. Dose is in mg/kg/day. The numbers in parentheses indicate the number of rats. c Average concentration differs between cortex and striatum ( p # 0.0083). * Different from controls (Turkey–Kramer test, a # 0.05). a b

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significance levels (critical a 5 0.025), indicating no significant effect of Aroclor 1254 on neurotransmitters or metabolites in striatal tissue. However, step-down analysis with no control of the false positive error rate suggested treatment-related changes in striatal DOPAC (F[2,13] 5 8.39, p 5 0.0046) and 5-HIAA (F[2,13] 5 8.15, p 5 0.0051) levels. Treatment with 10 or 30 mg/kg/day Aroclor 1254 tended to increase striatal DOPAC, and 30 mg/kg/day Aroclor 1254 slightly increased striatal 5-HIAA levels compared to controls. There were significant differences in the concentration of several of the neurotransmitters and/or their metabolites between the cortex and striatum (Table 1). Significant regional differences were indicated for NE, DA, DOPAC, HVA, and 5-HIAA (F’s[1,13] $ 63.30, p’s # 0.0001). The average concentrations of DA, DOPAC, HVA, and 5-HIAA were greater in the striatum than in the cortex. Conversely, the average concentration of NE was larger in the cortex than in the striatum (Table 1). Immunohistochemistry and Activity of Tyrosine Hydroxylase in Rats Repeatedly Dosed with Aroclor 1254 The effect of repeated administration of the PCB mixture Aroclor 1254 was evaluated by immunohistochemical staining for tyrosine hydroxylase, as well as tyrosine hydroxylase specific activity. This enzyme is involved in the biosynthesis of dopamine. There were no qualitative effects on tyrosine hydroxylase immunoreactivity in cell bodies of monaminergic nuclei in the hypothalamus, substantia nigra pars compacta, ventral tegmental area, or raphe nuclei (Fig. 5). In addition, no treatment-related changes in tyrosine hydroxylase immunoreactivity were observed in major monoaminergic fiber pathways for these neurons, such as the medial forebrain bundle, substantia nigra pars reticulata, and nigrostriatal pathway and striatum. Tyrosine hydroxylase specific activity, evaluated in minces of corpus striatum, was also not significantly different between control animals and animals dosed with 10 or 30 mg Aroclor 1254/kg/day for 4 weeks (Fig. 6), suggesting that dopamine synthesis is not significantly affected following repeated exposure to Aroclor 1254 in adult rats. Effects on Circulating Thyroid Hormone Levels PCBs were shown to cause hypothyroidism in prepubertal and developing animals (Byrne et al., 1987; Ness et al., 1993; Porterfield, 1994). In this study, we measured total and free T4 and T3 levels in serum samples to understand whether repeated exposure to Aroclor 1254 in adult rats is associated with alterations in circulating thyroid hormone levels. The ANOVA indicated a significant effect of treatment on total T4 (F[2,9] 5 287, p , 0.0001), free T4 (F[2,9] 5 54, p , 0.0001), total T3 (F[2,8] 5 5.3, p , 0.034), free T3 (F[2,9] 5 13.4, p , 0.002) levels. Circulating T4 (total and free) concentrations were drastically reduced following Aroclor 1254 treatment. The reduction was 95% in both 10 and 30 mg/kg dose groups (Fig.

7). Circulating T3 concentrations were also reduced following Aroclor 1254 treatment, however, the reductions were smaller compared to T4. The reduction in T3 concentrations were 29 to 42% in treated rats (Fig. 7). These results clearly suggest a severe hypothyroid state in rats treated with Aroclor 1254. Body Weight Gain Controlled Experiment Because PCB-exposed animals had a reduced body weight gain, we conducted weight-controlled experiments by restricting food to one group of rats (n 5 6) to match body weights of rats exposed to Aroclor 1254 for 4 weeks (see Fig. 1). Both behavioral and selected neurochemical experiments were conducted with these animals. In weight-controlled animals, horizontal (control, 4174 6 431; weight controlled, 4396 6 221 counts/30 min session), and vertical (control, 457 6 36; weight controlled, 428 6 37 counts/30-min session) motor activity were not different from normally fed animals. Similarly, calcium buffering by microsomes (control, 42.5 6 2.2; weightcontrolled, 48.4 6 1.6 pmol/mg protein/min) and mitochondria (control, 10.9 6 0.8; weight-controlled, 11.8 6 0.7 pmol/mg protein/min), as well as total PKC activity (control, 2.03 6 0.07; weight controlled, 2.37 6 0.16 nmol/mg protein/min) were not significantly different in weight-controlled rats compared to normal rats. Circulating T4 (control, 55.7 6 1.9; weight-controlled, 44.8 6 1.4 ng/ml serum; p , 0.0008) and T3 (control, 1.0 6 0.08; weight-controlled, 0.72 6 0.03 ng/ml serum; p , 0.0083) levels, however, were significantly lower (20 –28%) in weight-controlled rats compared to normal rats. These results indicate that behavioral and neurochemical changes observed during repeated exposure to Aroclor 1254 are not solely due to weight loss, but some of the decrease in circulating thyroid hormones by Aroclor 1254 could be attributed to weight loss. DISCUSSION

Some PCBs (laterally substituted, coplanar) and other halogenated hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-pdioxin, are generally believed to produce immunologic, teratogenic, reproductive, and carcinogenic effects by binding to the cytosolic aryl hydrocarbon (Ah) receptor (US EPA, 1991; Safe, 1994; Okey et al., 1994). However, neurotoxic effects of noncoplanar PCBs in adults as well as in developing animals do not appear to be mediated solely through the Ah receptor (Seegal, 1996). Seegal and his associates reported that orthosubstituted PCB congeners, which do not bind to Ah receptor, altered catecholamine levels in rat brains (Seegal and Shain, 1992; Seegal, 1994; Seegal and Schantz, 1994) and in cell cultures (Shain et al., 1991). Eriksson and Fredriksson (1996) reported that gestational exposure to lightly chlorinated orthosubstituted PCBs affect behavior of the offspring. Schantz et al. (1995) reported that perinatal exposure to ortho-substituted PCBs can result in long-lasting deficits in learning. However,

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FIG. 5. Tyrosine hydroxylase immunoreactivity in forebrain obtained from Long–Evans hooded rats orally administered Aroclor 1254 (0 or 30 mg/kg/day; 5 days/week) for 4 weeks. Immunoreactivity in sections of representative brain areas are shown for vehicle-treated control (A, C, and E) and treated (B, D, and F) rats. A and B are representative of the substantia nigra, C and D of the dorsal lateral quadrant of the striatum, and E and F of the hypothalamus. Arc, arcuate nucleus; mfb, medial forebrain bundle; PC, pars compacta; Pe, periventricular; PR, pars reticulata; Pa, paraventricular nucleus of the hypothalamus; VTA, ventral tegmental area; 3N, oculomotor nerve; 3V, third ventricle. Bar, 200 mm.

the mechanism by which these PCBs produce these neurobehavioral deficits is poorly understood. Previous research from our laboratory has indicated that second messenger systems including inositol phosphate, Ca21 homeostasis, and PKC translocation are also preferentially sensitive to ortho-substituted, noncoplanar PCBs in vitro (Kodavanti et al., 1993, 1994,

1995, 1996a,b; Shafer et al., 1996; Kodavanti and Tilson, 1997). Research from other laboratories also indicated that noncoplanar PCBs alter brain microsomal calcium transport (Wong et al., 1997). The present study was initiated to determine whether in vivo exposure at doses that would produce brain levels of PCBs in the range of those used in vitro would

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FIG. 6. Tyrosine hydroxylase activity in striatum obtained from Long– Evans hooded rats orally administered Aroclor 1254 (0, 10 or 30 mg/kg/day; 5 days/week) for 4 weeks. The enzyme activity in treated animals was not significantly different (p . 0.05) from that of control animals (4.9 6 0.4 pmol 14 CO2/mg protein/min). Data are expressed as means 6 SEM percent of control (n $ 4 animals). Samples from each rat were assayed in triplicate.

alter second messenger systems. The data indicate that intracellular Ca21 buffering and PKC can be affected in brain regions of rats exposed repeatedly with a commercial PCB mixture (Aroclor 1254) in the absence of changes in tyrosine hydroxylase levels or activity involved in dopamine biosynthesis. Rats exposed to 30 mg/kg showed decreased body weight gain and hypoactivity. Also, this exposure resulted in

accumulation of total PCBs in brain up to 15 ppm (Kodavanti et al., 1998), equivalent to 40 to 50 mM based on average molecular weight of 326.4 for Aroclor 1254 mixture (Erickson, 1986). The maintenance of intracellular Ca21 homeostasis is crucial for the normal growth and functioning of the neurons (Farber, 1990; Kater and Mills, 1991). Low levels (0.1 to 0.3 mM) of intraneuronal free Ca21 are maintained by the operation of Ca21 pumps located in plasma membrane, endoplasmic reticulum, and mitochondria (Carafoli, 1987). Cells must maintain a critical balance of buffering activities in order to maintain Ca21 homeostasis and function normally (Wang et al., 1997). A significant alteration in these processes may perturb Ca21 homeostasis and trigger the events that lead to altered neuronal growth and cytotoxicity (Nicotera et al., 1992). We previously showed that ortho-substituted PCBs can inhibit microsomal and mitochondrial Ca21 sequestration at low micromolar (IC50 5 2–15 mM) concentrations in vitro (Kodavanti et al., 1996a). Present in vivo data indicate that Ca21 sequestration by microsomes and mitochondria are also affected following repeated exposure to Aroclor 1254. Of the two organelles, microsomes are more sensitive to PCB effects than mitochondria. For example, only microsomal Ca21 uptake is affected in frontal cortex and striatum. This effect could be due to differential accumulation of PCBs intracellularly, since other halo-

FIG. 7. Circulating thyroid hormone levels (T4 and T3) in rats following exposure to Aroclor 1254 (0, 10, or 30 mg/kg/day) for 4 weeks (5 days/week). Values are means 6 SEM of four rats. Samples from each rat were assayed in duplicate and an average value was obtained for each rat. *Significantly different from control at p , 0.05.

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genated hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-pdioxin (Santostefano et al., 1996) and 2,3,4,7,8-pentachlorodibenzofuran (Yoshimura et al., 1987) accumulate preferentially in liver microsomal fraction over cytosolic and mitochondrial fractions. Within the brain regions examined for this end point, cerebellum seems to be more sensitive for PCB effects than frontal cortex or striatum. There are several environmental chemicals that are known neurotoxicants, including chlordecone (Desaiah et al., 1985), triorganotins (Prasada Rao et al., 1985), lead (Silbergeld and Adler, 1978), and mercury (Binah et al., 1978), have been reported to inhibit Ca21 sequestration by intracellular organelles and/or Ca21–ATPase involved in Ca21-extrusion process. The present data suggest that intracellular calcium buffering is a sensitive endpoint for noncoplanar PCBs accumulated in brain and may be involved in the neurotoxicity of PCBs. PKC is a family of ubiquitous phospholipid-dependent serine/threonine kinases, which play key roles in cellular signal transduction (Nishizuka, 1992) and are involved in a variety of physiological and toxicological phenomena (Mattson, 1991; Eboli et al., 1993; Felipo et al., 1993). PKC is activated and also translocated from cytosol to the membrane by several factors including a rise in intracellular free calcium (Trilivas and Brown, 1989). Previous studies in cerebellar granule cell cultures indicate that acute exposure to PCBs (starting at 10 mM) increases PKC translocation and this effect is dependent on the presence of external Ca21 (Kodavanti et al., 1995). Consistent with the in vitro studies, current studies showed that Aroclor 1254 significantly increased membrane-associated PKC activity in cerebellum. Aroclor 1254 also significantly decreased total PKC activity, which may reflect down-regulation of the enzyme after persistent stimulation (Favaron et al., 1990). The changes in PKC activity seen in cerebellum were not observed in any other brain region studied, indicating brain-region specific effects of PCBs on PKC. The brainregion specific effect may be partially related to differential accumulation/distribution of various PCB congeners in these three brain regions. Total PCBs in cerebellum, frontal cortex, and striatum were 13.1, 15.1, and 8.2 ppm, respectively (Kodavanti et al., 1998; companion paper). In general, these results are in agreement with results on calcium buffering and suggest that PKC is also a sensitive endpoint, at least in some brain regions, for PCBs in vivo, as it is known to be in vitro. Further studies focused on other proposed neurochemical endpoints of PCB effects. The analysis of neurotransmitter levels in rats exposed to Aroclor 1254 indicated that there were no differences in DA, NE, 5-HT or HVA levels in cortex or striatum in treated animals compared to controls. However, small increases in striatal DOPAC and 5-HIAA were observed following Aroclor 1254 exposure. The lack of effects of Aroclor 1254 on biogenic amine levels in the cortex agrees with previous studies that indicated that rats fed Aroclor 1254 adulterated food (500 and 1000 ppm) did not have altered levels of NE and 5-HT when compared to controls (Seegal et

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al., 1991a). However, lack of effects on striatal DA levels is not in agreement with previous reports (Seegal et al., 1991a) where DA and its metabolites were significantly decreased in striatum of rats fed Aroclor 1254 adulterated food (500 and 1000 ppm). This discrepancy could be explained by the differential accumulation of PCB congeners in striatum with the two protocols, since Seegal et al. (1991a) reported accumulation of total PCBs of 41.6 mg/g, whereas our toxicokinetic data indicated only 8.24 mg/g of total PCBs in striatum (Kodavanti et al., 1998). The lack of a significant overall treatment effect in the present study suggests that the minor changes in DOPAC and 5-HIAA in the striatum may represent a Type I statistical error. Statistical analysis indicated that there was significant power to detect regional differences in neurotransmitter and metabolite levels. This suggests that the lack of change in neurotransmitter levels was not due to the lack of statistical power. Thyroid hormones play a crucial role in brain function (Ford and Cramer, 1977; Porterfield and Hendrich, 1993) and PCBs and related chemicals reduce circulating T4 and T3 levels (Byrne et al., 1987; Ness et al., 1993; Morse et al., 1993, 1996; Porterfield, 1994). Our results are in agreement with these reports where repeated exposure to Aroclor 1254 resulted in 95% drop in circulating total and free T4 levels, indicating PCBs cause a hypothyroid state in adults as well as in animals during development. The preferential effect of PCBs on serum T4 levels could have neurological implications because 80% of the thyroid hormones in the brain come from serum T4 (Silva and Matthews, 1984). In summary, these results indicate that in vivo exposure to a PCB mixture can produce neurochemical changes related to intracellular Ca21 buffering and PKC, which are similar to those observed in vitro in neuronal cell cultures. Repeated exposure of adult rats to Aroclor 1254 caused changes in brain second messengers in the absence of changes in neurotransmitters or tyrosine hydroxylase. Higher doses of Aroclor 1254 cause hypoactivity in rats. The doses used (10 to 30 mg/kg/ day) are relatively high compared to the levels found in the environment. However, PCBs are ubiquitous compounds and bioaccumulate due to low degradation, and humans could be exposed from a number of sources, including drinking water and food products (fish, meat, and dairy products). In selected areas, the levels of PCBs in drinking water ranged from 100 to 450 ng/liter and in food products it was over 200 mg/kg fresh weight (WHO, 1993). Bush and Kadlec (1995) have also reported that zebra mussels from Niagara River have PCB levels in the mg/kg range. In humans, levels of PCBs in the adipose tissue of occupationally exposed workers ranged from 2.2 to 290 ppm (WHO, 1993), whereas adipose tissue of rats dosed with Aroclor 1254 in the present study contained PCB levels of 552 ppm (Kodavanti et al., 1998). Similarly, blood concentrations of PCBs detected in capacitor-manufacturing workers were as high as 3.5 mg/ml (Wolff, 1985), whereas rats in the present study contained blood levels of 1.5 mg/ml

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(Kodavanti et al., 1998). Based on this information, doses that affected neurobehavior or altered cellular second messenger systems are not inconsistent with those found in the environment or in occupational workers. Additional studies are needed to extrapolate this information to the observed neurobehavioral changes following repeated exposure to low levels of PCBs. ACKNOWLEDGMENTS The authors thank Mr. Jackie Farmer for his help in dosing animals and testing the animals for motor activity, Ms. Jamie Graff for her technical assistance in neurotransmitter analysis, and Ms. Najwa Haykal-Coates for her technical assistance with the neuroanatomical assays for tyrosine hydroxylase. Authors also thank Drs. Susan Schantz and Dennis Morse for their critical comments on an earlier version of this manuscript.

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