Polychlorinated Biphenyls (PCBs) Selectively Disrupt Serotonergic

50, 54 – 63 (1999) Copyright © 1999 by the Society of Toxicology

TOXICOLOGICAL SCIENCES

Polychlorinated Biphenyls (PCBs) Selectively Disrupt Serotonergic Cell Growth in the Developing Spisula Embryo Cynthia R. Smith,* Colin M. Barker,* Lewellys F. Barker,† Kathryn Jessen-Eller,* and Carol L. Reinisch* ,‡ ,1 *Department of Environmental and Population Health, Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536; †Laboratory of Marine Biomedicine, Visiting Scientist, Marine Biological Laboratory, Woods Hole, Massachusetts 02543; and ‡Laboratory of Marine Biomedicine, Marine Biological Laboratory, Woods Hole, Massachusetts 02543 Received May 18, 1998; accepted November 18, 1998

utero. These deficits include learning disorders, intellectual impairment (Goldman-Rahic, 1991; Jacobson and Jacobson, 1996), short-term memory loss, attention disorder (Jacobson et al., 1990), and hypotonic and hyporeflexic motor dysfunction (Gladen and Rogan, 1991; Rogan et al., 1988). Based on evidence of a direct link between PCB exposure and neuronal impairment, our laboratory focused on identifying pathways of PCB-induced modulation of neuronal growth in the developing embryo. The present study evaluated how exposure of embryos to environmentally relevant doses of a PCB mixture, Aroclor 1254, altered serotonergic cell growth. Our approach utilized a novel, marine invertebrate embryo model designed to quantitate subtle mechanistic changes in early neuronal development. The surf clam, Spisula solidissima, was specifically chosen for the unique synchronicity of embryonic development within this species (Dessev and Goldman, 1990; Hunt et al., 1992; Longo et al., 1994). Furthermore, neuronal growth in Spisula is rapid and quantifiable during early embryonic development. To evaluate neuronal growth, we chose serotonin as a biomarker of early nervous system development. Serotonin, a highly conserved neurotransmitter, is critically important because of its pivotal role in regulating neuronal growth, differentiation, and plasticity during embryogenesis in vertebrate and invertebrate animals (Benton, 1994; Lauder, 1982; Whitaker-Azmitia, 1991). Serotonergic cells are among the first categories of neurons to differentiate and to express their transmitter phenotype on the developing nervous system (Lidov and Molliver, 1982). Furthermore, involvement in nervous system development, learning behaviors, and memory (Kravitz, 1988; Willows, 1985) make serotonin a potential target of nervous system disruption by PCBs. Therefore, we tested the hypothesis that PCBs selectively impair serotonergic cell development during embryogenesis.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that exert neurotoxic effects during embryonic development. The present study demonstrates that early embryonic exposure to a mixture of PCBs (Aroclor 1254) results in a decrease in serotonergic cell growth. Using a novel, marine invertebrate embryo model, Spisula solidissima, immunocytochemistry, and confocal microscopy techniques, a dose-dependent decrease in serotonergic cell number was quantified within 24 h of exposure. This effect was seen with doses as low as 1 ppm Aroclor 1254. These findings demonstrate that environmentally relevant doses of Aroclor 1254 impair development of the serotonergic nervous system. Key Words: PCBs; environmental contamination; invertebrate embryo model; Aroclor 1254; Spisula solidissima.

Polychlorinated biphenyls (PCBs) are a family of 209 chemical congeners with varying degrees of chlorination. PCBs were designed as highly stable industrial compounds and have been used primarily as heat transfer fluids, dielectric fluids, and hydraulic lubricants (Safe, 1992). The accidental and deliberate dumping of these chemicals has led to global contamination and bioaccumulation of PCBs within the food chain (Seiler et al., 1994). PCBs are found in almost every natural medium including water, air, and soil, as well as human and animal tissues (Kimbrough, 1995; Kimbrough and Jensen, 1989). Despite widespread environmental contamination of these toxicants, their adverse health effects are only partially understood. Both experimental and epidemiological data suggest that PCBs disrupt embryonic development of the nervous system at environmentally relevant doses. Laboratory data in rats and non-human primates have correlated prenatal and perinatal PCB exposure to deficits in spatial learning, memory, neuronal morphology, and intellectual function (Levin et al., 1988; Shantz et al., 1992). In addition, epidemiological studies have described neuronal deficits in children exposed to PCBs in

MATERIALS AND METHODS

1 To whom correspondence should be addressed at Laboratory of Marine Biomedicine, 7 MBL Street, Marine Biological Laboratory, Woods Hole, MA 02543. Fax: (508) 540-6902. E-mail: [email protected].

The marine invertebrate embryo model. Adult surf clams, Spisula solidissima, were collected by the Marine Biological Laboratory (Woods Hole, MA) from Menemsha Bay, Martha’s Vineyard, MA. Clams were stored at 18°C in 54

PCBS DISRUPT SEROTONERGIC CELL GROWTH continuously-running sea water. All experiments were carried out in artificial seawater, so that no naturally occurring contaminants were present. Artificial seawater parameters were set as follows: pH 8.0, 30 ppt salinity, 18°C. In addition, all experiments described were conducted in a single-blinded manner. In vitro fertilization. Following the methods of Allen and Clotteau with moderate modifications (Allen, 1953; Clotteau and Dube, 1993; Hunt et al., 1992; Loosanoff, 1954; Loosanoff and Davis, 1963), in vitro fertilization of the surf clam, Spisula solidissima, was performed. Briefly, gametes were excised from adult Spisula and the oocytes were washed 3 times in artificial seawater. Gametes were combined at a sperm to egg ratio of 100:1 for 10 min with continuous stirring in 100 ml of artificial seawater. Oocytes were allowed to settle for 8 min, after which time the sperm and supernatant were siphoned off. The volume was readjusted and the fertilization volume divided into 6 100-mm petri dishes. Each dish contained approximately 10,000 embryos. Exposure of developing embryos to PCB. Embryos were exposed to environmentally relevant doses of Aroclor 1254 (Harper et al., 1994; Lake et al., 1992; Mes, 1990), a mixture of coplanar and ortho-substituted noncoplanar PCBs containing 132 congeners and 54% chlorine by weight (Safe, 1992). Aroclor 1254 (Ultra Scientific, North Kingstown, RI) was selected because it is a predominant mixture of PCBs in New Bedford Harbor, Massachusetts, where a high prevalence of leukemia has been documented in a related molluscan species (Barker et al., 1997; Miosky et al., 1989; Reinisch et al., 1984). Aroclor 1254 was dissolved in acetone and diluted in artificial seawater. Aroclor 1254 solutions (1, 10, 100, or 500 ppm) were added to fertilized oocytes immediately following germinal vesicle breakdown, to study the effects of post-fertilization exposure. Acetone and artificial seawater (0 ppm) were added to parallel populations of fertilized oocytes as controls. Model validation and time course evaluation. To test the reproducibility of the model, 4 primary post-fertilization events were recorded (polar body extrusion, pronuclei formation, first cleavage, and second cleavage) and their time course plotted for each contol and exposed embryo sample. Normal time courses were collected from 9 in vitro fertilization experiments. In addition, time course experiments were conducted in triplicate for each Aroclor 1254 exposure concentration and acetone vehicle control, and then duplicated over 2 consecutive summers (seasonal breeding period for Spisula). In order to focus on post-fertilization events, oocytes that had not been successfully inseminated were omitted from the study prior to exposure. In addition, Spisula oocytes do not have protective mechanisms against polyspermy, so experiments were aborted if polyspermy was observed. Polyspermy was recognized as the formation of 3 or more pronuclei in the fertilized oocyte. Although this event can not be detected until pronuclei formation, polyspermy actually occurs at the time of insemination (time zero). Aroclor 1254 was only added following the successful fertilization of oocytes, detected by germinal vesicle breakdown (15 min post-fertilization). Therefore, Aroclor 1254 was not present during the insemination event and could not influence the presence of polyspermy. Embryo wash. At 5 h post-fertilization, corresponding to the 8-cell stage of embryonic development, all Aroclor 1254 and control solutions were removed. The 5-h time period for exposure was chosen because critical stages of early embryonic development occur during this interval. Following removal of the contaminant and control solutions, embryos were washed 4 times in artificial seawater. Embryos were transferred to 4-L tanks at 6 h post-fertilization and maintained at 18°C in artificial seawater. Immunofluorescence technique for labeling serotonin. Immunofluorescence techniques were used to identify serotonergic cells as follows. Samples of embryos were taken at 24, 48, and 72 h post-fertilization and immediately fixed in 4% formaldehyde for 1 h at room temperature. Embryos were aliquoted into 1-ml samples on ice and gently spun at 1250 g (4°C) for 10 min in a Beckman Ultracentrifuge (Beckman Instruments, Inc., Carlsbad, CA). The supernatant was siphoned off, and the pellet was resuspended in artificial seawater and allowed to soak for 10 min. This washing procedure was repeated 3 times. Rabbit serotonin antisera (Incstar, Stillwater, MN) and normal rabbit serum (Boehringer-Mannheim, Indianapolis, IN) were each diluted to 1:2000

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in a PBS/0.3% Triton X-100 solution, then centrifuged at 14,000 g for 10 min at 4°C. Each embryo sample was divided into 2 groups and incubated in either serotonin antiserum or normal rabbit serum for approximately 16 h at 4°C. All groups were subsequently washed 3 times and incubated with a 1:100 dilution of goat anti-rabbit immunoglobulin labeled with the Oregon Green fluorophore (Molecular Probes, Eugene, OR) in artificial seawater for 1 h at 18°C. The samples were then washed 3 times and stored at 4°C overnight. Dual immunofluorescence technique for labeling PCB. Following immunofluorescent labeling for serotonin, embryos were incubated with a second antibody raised against PCB. A purified chicken anti-PCB immunoglobulin (Maine Biotechnology Services, Portland, ME) was selected for its affinity to Aroclor 1254. (This antibody also binds to Aroclors 1016, 1242, 1248, 1260, and 1268.) Embryos were incubated in either a 1:1000 dilution of chicken anti-PCB immunoglobulin in a PBS/0.3% Triton X-100 solution or a 1:10,000 dilution of normal chicken serum (Jackson Immunologics, West Grove, PA) in a PBS/0.3% Triton X-100 solution for approximately 16 h at 4°C. Embryos were washed 3 times, and donkey anti-chicken immunoglobin conjugated to Cy3 (Jackson Immunologics, West Grove, PA) diluted 1:250 in artificial sea water was added to all embryos for 1 h at room temperature. Finally, embryos were washed as previously described. Detection of DNA by DAPI fluorescence. Embryos were transferred into a 0.002 mg/ml DAPI (4,6-diamidino-2-phenylindole dihydrochloride) solution in 25% glycerol/75% artificial seawater and prepared as whole mounts for microscopy. DAPI (Sigma, St. Louis, MO), a fluorescent dye that binds to DNA, was used to identify cell nuclei, allowing for detection and quantitation of fluorescent DNA emitted from each embryo. Embryos were divided into duplicate slide sets, one to be used for DAPI fluorescence quantitation and the other for confocal microscopy. Embryo size measurement and DNA detection. Embryo size, measured as area, was evaluated simultaneously with DNA content, measured as DAPI fluorescence. These two parameters were used to compare the embryonic stage of each exposure group by quantitating embryo sizes and the amount of fluorescent DNA emitted from each embryo. In both cases, an image of the embryo was captured with a digitizing camera (Spot, Diagnostic Instruments, Inc., Sterling Heights, MI) and a Zeiss Axiophot fluorescent microscope (Carl Zeiss, Inc., Thornwood, NY), and analyzed using the Metamorph Imaging System (Universal Imaging Corp., West Chester, PA). The transmitted image was captured under automated exposure conditions for red, green, and blue light. DAPI fluorescence was visualized and imaged in embryos at a magnification of 4003. The DAPI image was limited to blue light and an exposure time of 2 s. Transmitted and DAPI imaging was performed on groups of 5–13 embryos per exposure level. Dual imaging of serotonergic cells and Aroclor 1254 by confocal microscopy. Embryos were imaged on a Zeiss Confocal 410 Laser Scanning Microscope (Carl Zeiss, Inc., Thornwood, NY) at the Central Microscopy Facility of the Marine Biological Laboratory, based upon emerging confocal microscopy techniques (Ettensohn and Malinda, 1993; Ploem, 1987; Shotton, 1989). Dual imaging of serotonergic cell bodies by the Oregon Green fluorophore (laser, 488; emission, 515) and Aroclor 1254 distribution by the Cy3 fluorophore (laser, 543; emission, 570) was performed. A 253 water immersion lens was used and the imaging parameters were set as follows: magnification, 253/0.8; zoom, 3.282; pinhole, 12; time, 9.04 s. Quantitation of serotonergic cells by confocal microscopy. Following image aquisition of individual embryos, sequential 1 micron sections (z series) were used to quantitate serotonergic cells in fixed embryos at 24, 48, and 72 h post-fertilization. Three-dimensional analyses were used to reconstruct and rotate embryos to confirm the number of serotonergic cells counted. Confocal microscopy experiments were performed in duplicate with 4 –7 embryos/ exposure level per experiment. Statistical analyses. All experiments were conducted in a single-blinded manner. Fertilization time course data were statistically analyzed by single factor ANOVA. DAPI imaging data was assessed by ANOVA analysis on

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FIG. 2. Time course of post-fertilization events. These data represent the average time points at which exposed and unexposed embryos completed each post-fertilization event.

FIG. 1. Initial fertilization events of the Spisula embryo: (A) polar body extrusion; (B) pronuclei formation; (C) first cleavage; (D) second cleavage. These 4 events were recognized and recorded for fertilization time course analyses.

mean embryo size and total fluorescence emitted. Serotonergic cell numbers were analyzed with chi-square testing and regression analyses.

RESULTS

In vitro Fertilization Time Course Studies Our first step was to determine the reproducibility of the Spisula model by plotting time courses of initial post-fertilization events. First, an artificial seawater control time course of 4 post-fertilization events (polar body extrusion, pronuclei formation, first cleavage, and second cleavage; Fig. 1) was determined from nine in vitro fertilization experiments (Table 1). Second, time courses were recorded for each Aroclor 1254

exposure level and acetone vehicle control, and compared to the artificial sea water control time course (Fig. 2). The results demonstrated that the time courses were reproducible and did not significantly vary between experiments. Furthermore, the Aroclor 1254 exposure time course and the acetone vehicle control time course showed no delay in relation to the artificial sea water control (0 ppm) time course (p $ 0.27). Therefore, neither the acetone vehicle control nor Aroclor 1254 significantly delayed the completion of these four initial post-fertilization events. Comparison of Embryonic Stage of Development between Exposure Groups Our second objective was to evaluate whether Aroclor 1254 modified development of the entire embryo by 24 h post-fertilization. To confirm the developmental stage of each embryonic group, we quantitated embryo size and embryonic DNA content and compared these values among exposed and unexposed embryos. First, embryo area was determined by sketching the perimeter of each transmitted

TABLE 1 Normal Time Course of Post-Fertilization Events for Spisula Embryos Event

Time (min)

Fertilization Germinal vesicle breakdown Polar body extrusion Pronuclei formation First cleavage Second cleavage

0 15 50 6 9 72 6 8 112 6 9 173 6 18

Note. These data represent the average time points (6 standard deviation; n, 12) at which 100% of the embryos observed were at a particular stage of fertilization. Refer to Figure 1 for images of embryos at the following stages: polar body extrusion, pronuclei formation, first cleavage, and second cleavage.

TABLE 2 Average Number of Serotonergic Cell Bodies per Embryo Aroclor 1254

24 h 48 h 72 h

0 ppm

1 ppm

10 ppm

100 ppm

500 ppm

1.30 6 0.15 n, 10 4.09 6 0.21 n, 11 3.80 6 0.13 n, 10

1.08 6 0.15 n, 12 3.50 6 0.15 n, 12 3.60 6 0.16 n, 10

1.00 6 0.00 n, 12 3.42 6 0.15 n, 12 3.22 6 0.15 n, 9

1.00 6 0.12 n, 12 3.00 6 0.20 n, 13 3.09 6 0.21 n, 11

0.56 6 0.18 n, 9 3.00 6 0.16 n, 13 3.00 6 0.15 n, 10

Note. Embryos have been exposed to a range of Aroclor 1254 concentrations (0 ppm–500 ppm) and have been evaluated at 24, 48, and 72 h post-insemination.

PCBS DISRUPT SEROTONERGIC CELL GROWTH

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FIG. 3. Average size of exposed and unexposed embryos, measured by sketching the perimeter of each transmitted light image and then calculating the embryo area. Error bars represent standard deviation.

light image (Fig. 3). The results showed no significant difference (at p # 0.05) in mean embryo area across the experimental groups (acetone vehicle control, 0, 1, 10, 100, and 500 ppm Aroclor 1254). Second, the DAPI image was analyzed for total DNA content by quantitating total fluorescence emitted by each embryo (Fig. 4). Total DNA content did not significantly vary (at p # 0.05) across the experimental groups. These data indicated that all embryos were approximately the same size and did not differ in total DNA content. Therefore, Aroclor 1254 did not delay development of the entire embryo. Next, we investigated whether or not PCBs selectively altered the serotonergic system during the early events of embryogenesis.

FIG. 4. Total DNA content of exposed and unexposed embryos. DAPI, a fluorescent DNA dye, was used to determine total DNA content of exposed and unexposed embryos. For each embryo, the total amount of fluorescent light emitted from the DAPI dye was quantitated. Error bars represent standard deviation.

FIG. 5. Average numbers of serotonergic cell bodies in treated and untreated embryos. Error bars represent standard deviation.

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59

FIG. 6. Confocal microscopy images of exposed and unexposed embryos at 24, 48, and 72 h post-fertilization; 6a, 6b, and 6c show transmitted images and 1-micron cross-sectional fluorescent images of representative embryos exposed to 0, 1, 10, 100, or 500 ppm Aroclor 1254, as well as a control embryo for the antibody (normal rabbit serum or normal chicken serum). Figure 6a shows images of 24-h post-imsemination embryos; 6b shows 48-h embryos, and 6c shows 72-h embryos. Serotonin localization as detected by green fluorescence (Oregon green fluorophore) allowed us to quantitate the number of serotonergic cell bodies in these embryos. Aroclor 1254 distribution as detected by red fluorescence (Cy3 fluorophore) allowed us to visualize Aroclor 1254 sequestration in the embryos. Each column in Figure 6a contains 2 images of one representative embryo. The transmitted light image of the embryo is in the top row and a corresponding cross-sectional fluorescent image (1 micron thick) is in the bottom row. In 6b and 6c, 2 fluorescent images of each embryo were included (middle row and bottom row) if more than one cross section was needed to demonstrate the total number of serotonergic cell bodies quantitated. (Images J and K in 6b are an exception; the neurons observed in K are a continuation of the neurons observed in J.)

Quantitation of Serotonergic Cells in the Developing Embryo A serotonin polyclonal antibody was used to identify and quantitate serotonergic cell bodies in developing embryos utilizing confocal microscopy techniques. Specifically, we quantitated the number of serotonergic cells formed within embryos at 24, 48, and 72 h post-fertilization, and then compared the number of serotonergic cells between exposed and unexposed embryos. Our results demonstrated that embryos exposed to 1, 10, 100 and 500 ppm Aroclor 1254 showed a dose-dependent decrease in serotonergic cell number when compared to the acetone and artificial sea water (0 ppm) control embryos (Table 2; Figs. 5, 6). This dose-dependent decrease was detectable as early as 24 h post-fertilization, and was also seen at 48 and 72 h post-fertilization. Statistical analyses confirmed significant relationships between increasing Aroclor 1254 concentrations and decreasing numbers of serotonergic cell bodies (p # 0.002, 0.003, and 0.002, respectively, for 24, 48, and 72 h). Regres-

sion analysis confirmed this relationship by showing a highly significant reduction of approximately 0.1 neurons per log unit of Aroclor 1254 concentration (p # 0.001) at each time point (24, 48, and 72 h). Therefore, these experiments confirmed that exposure to Aroclor 1254 significantly decreased the number of serotonergic cells in developing Spisula embryos. Next, we assessed whether or not the loss of serotonergic cells was due to the direct uptake of Aroclor 1254 within serotonergic cell bodies. Evaluation of Concurrent PCB Distribution in Serotonergic Cells To investigate the potential relationship between Aroclor 1254 exposure and serotonergic cell loss, we evaluated embryos from each exposure group to determine if serotonergic cell bodies also contained Aroclor 1254. The data showed PCB antibody accumulation in embryos treated with 1, 10, 100, and 500 ppm Aroclor 1254 at 24, 48, and 72 h post-fertilization.

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FIG. 7. Localization of PCB antibody in a non-serotonergic cell body: this 72 h post-fertilization clam embryo was exposed to 100 ppm Aroclor 1254. The embryo was incubated with both a serotonin antibody (green) and a PCB antibody (red). (A) is the transmitted image and (B) is a 1-micron cross-sectional confocal image. In (B), the PCB antibody has localized in a non-serotonergic cell body, and the serotonin antibody has localized in a serotonergic cell body.

PCB antibody was found diffusely distributed throughout the entire embryo (Fig. 6). In addition, PCB antibody was also found within selected cell bodies (Fig. 7). However, no embryos demonstrated concurrent localization of PCB antibody and serotonin antibody within the same cell bodies.

DISCUSSION

The purpose of this study was to determine if exposure of developing embryos to environmentally relevant levels of Aroclor 1254 modified nervous system development. First, Aroclor 1254 exposure, regardless of concentration, did not delay initial post-fertilization events or the development of the entire embryo. Second, serotonergic cells within developing embryos were decreased in a dose-dependent manner, due to early exposure of embryos to Aroclor 1254, which provides strong evidence that serotonergic cell loss was chemically-induced. Therefore, Aroclor 1254 decreased the number of serotonergic cells in the developing nervous system rather than delaying development of the whole embryo. Given that Aroclor 1254 preferentially targeted the serotonergic system rather than the entire embryo, we predicted that Aroclor 1254 would be found in the serotonergic cells of exposed embryos. However, sequestration of Aroclor 1254 did not occur in serotonergic cells, but instead was localized within non-serotonergic cell bodies as well as diffusely distributed throughout the developing embryo. Labeling of non-serotonergic cells with an antibody to PCB raised numerous questions regarding the identity of those cells. A principle candidate is the dopaminergic cell. Dopamine is another highly conserved neurotransmitter in vertebrate and invertebrate animals (Cournil, 1995), and studies have shown that PCBs directly disrupt the dopaminergic system (Seegal and Chishti, 1995; Chishti et

al., 1996). Studies directed toward identifying these cells currently are underway in our laboratory. The data presented in this paper support previously published work demonstrating that PCBs alter neurotransmitter levels and nervous system function. Table 3 references research that has correlated PCB exposure to nervous system abnormalities in children, non-human primates, rats, and fish. The list is a cross section of the work done in this area, and illustrates the range of doses (0.8 – 1000 ppm) and various routes of exposure (oral, transplacental, lactational) that have been used to study the neurotoxicity of PCBs. Specifically, several of these studies have shown PCBs to decrease levels of serotonin in the central nervous system, as well as increase levels of serotonin metabolites. Our study supports this data, and therefore offers a reliable method of rapidly detecting subtle changes in the development of the serotonergic nervous system. The most important finding of this study was that environmentally relevant doses of Aroclor 1254 (1–500 ppm) impaired the serotonergic system in the developing embryo. This data may explain the correlation that has been documented between in utero PCB exposure and intellectual impairment in vertebrates. For example, previous studies have shown that serotonergic disruption during embryogenesis can delay nervous system development by interfering with neuronal growth, differentiation, maturation, and synapse formation (Lauder, 1983, 1990). Furthermore, serotonergic alteration may have profound effects on learning and memory. A recent study by Mazer et al. (1997) reported that experimental depletion of serotonin with parachlorophenylalanine (a tryptophan hydroxylase inhibitor) during synaptogenesis led to neurodevelopmental disorders, including learning deficits in the adult rat. Serotonin reduction also led to long-term alterations in neuronal morphology and function, and interfered with spatial learning and memory

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TABLE 3 Previous Studies Conducted with PCB Compounds Demonstrating Nervous System Abnormalities Species Rats Nonhuman primates

Compound(s)

Exposure, dose

Results

Aroclor 1254, Aroclor 1260 Aroclor 1016, Aroclor 1260

Oral gavage (500 mg/kg, 1000 mg/kg) Oral ingestion, (0.8–3.2 mg/kg/day) for 20 weeks Oral ingestion (500 mg/kg, 1000 mg/kg) In utero, lactational

Alteration of brain serotonin levels and its metabolites Decrease in central dopamine levels and its metabolites

Seegal, 1986

Decrease in brain dopamine levels and metabolism Deficits in spatial learning and memory Increase in serotonin metabolism seen in offspring Deficits in short-term memory, attention, intellectual function Decrease in serotonin and dopamine levels; increased metabolism

Seegal, 1991b

Rats

Aroclor 1254

Nonhuman primates

Aroclor 1248

Rats (90 days post-natal)

Aroclor 1254

Pregnant rats received oral gavage (5, 25 mg/kg)

Children

Aroclor 1260

Transplacental, lactational

Fish

Aroclor 1254

Oral ingestion (0.1 mg/100 g body wt/day)

systems. Therefore, PCB-induced disruption of the serotonin pathway during nervous system development may lead to permanent intellectual impairment. To explain the neurotoxic effect of PCBs on the serotonergic system, we have considered three possible mechanisms. PCBs could (1) decrease synthesis, (2) alter release, and/or (3) increase metabolism of serotonin. First, PCBs could decrease serotonin synthesis by interfering with tryptophan hydroxylase, the rate-limiting enzyme in the serotonin biosynthesis pathway. This hypothesis is based on evidence that non-coplanar PCBs (found in Aroclor 1254) reduce the activity of tyrosine hydroxylase, a structurally- and functionally-related enzyme (Seegal, 1994). Tyrosine hydroxylase is the rate-limiting enzyme for the production of dopamine (Levitt et al., 1965). A PCB-induced decrease in tyrosine hydroxylase activity leads to a reduction of dopamine synthesis in the brain (Chishti and Seegal, 1992; Seegal and Chishti, 1995). Similarly, non-coplanar PCBs may interfere with tryptophan hydroxylase activity, and therefore account for a decrease in serotonin synthesis. Second, PCBs could alter the release of serotonin from developing neurons by interrupting the function of serotonin as an inducer of neuronal growth. Recent publications have shown that PCB congeners alter neuronal calcium homeostasis through activation of the ryanodine receptor, which is a receptor that regulates the release of calcium from endoplasmic reticulum (Wong et al., 1997; Wong and Pessah, 1996). PCB congeners maintain the ryanodine receptor in an active state, causing an increase in intracellular calcium. This could potentially interfere with calcium-dependent cellular processes, such as serotonin release and neuronal transmission. In fact, blockade of the ryanodine receptor during early neuronal development alters seroto-

Reference

Seegal, 1991a

Schantz, 1992 Morse, 1996

Jacobson, 1996

Khan, 1996

nergic growth in Spisula embryos by increasing neurite outgrowth (Jessen-Eller et al., in press). This suggests that a PCB-induced activation of the ryanodine receptor may also disrupt intracellular calcium levels and lead to a decrease in serotonergic cells. Therefore, PCB-induced disruption of cellular calcium levels may lead to a decrease in serotonin release from cell bodies. The third possible mechanism is a PCB-induced increase in serotonin metabolism. Studies have shown PCB mixtures, particularly Aroclor 1254, increase serotonin metabolite concentrations in the brain (Seegal et al., 1986). A recent publication by Morse et al. (1996) demonstrated an increase in serotonin turnover in rodents that had been exposed to Aroclor 1254 in utero and suggested a long-term alteration in the serotonergic system. Similarly, a study conducted in fish demonstrated that the ingestion of Aroclor 1254 resulted in a decrease in serotonin levels and an increase in the ratio of serotonin metabolite to serotonin (Khan and Thomas, 1996). These studies suggest that PCB exposure results in an overall increase in serotonin metabolism, particularly in the central nervous system. Further study of the interaction between PCBs and the serotonin pathway must be conducted to continue to define the mechanism of neurotoxicity. ACKNOWLEDGMENTS The authors would like to thank Dr. Will Rand for statistical analyses of the data, Dr. Mark Martindale for his expert advice on embryo imaging techniques, and Dr. Joan King for her contributions to the development of microscopic quantitation techniques. In addition, we appreciate the assistance of Dr. Roxanna Smolowitz in the histological evaluation of clam gametes, Louie Kerr in the acquisition of confocal microscopy images, Robert Brown for the contribution of computer graphic expertise, and Dr. Robert Palazzo for his expertise

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on Spisula fertilization. This work was supported, in part, by National Institutes of Health Grants T35DK07635 and RO1-CA44307 and by grants from the Sweet Water Trust and the Geraldine R. Dodge Foundation.

dechlorination processes and pathways in the New Bedford Harbor sediments. Marine Environ. Res. 33, 31– 47. Lauder, J. M. (1983). Hormonal and humoral influences on brain development. Psychoneuroendocrinology 8, 121–155.

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