Polychlorinated Biphenyls (PCBs) Disrupt Development of the Serotonergic Nervous System in the Surf Clam Embryo
Abstract
The potential toxicity of polychlorinated biphenyls (PCBs) on aquatic animal health has been a topic of widespread controversy for decades. PCBs were designed as highly stable industrial compounds, capable of withstanding extreme temperatures and treatments. PCBs have been primarily used as heat transfer fluids, dielectric fluids, and hydraulic lubricants.9 Although PCBs have been banned by the U.S. Environmental Protection Agency since 1977, the continued accidental and deliberate dumping of these chemicals has led to global contamination and bioaccumulation of PCBs within the food chain.11 Recent studies have shown PCBs to be potential neurotoxic agents. Specifically, both experimental and epidemiological data suggest that PCBs disrupt embryonic development of the nervous system at environmentally relevant doses.3,5,10 The goal of this study was to determine how exposure of embryos to Aroclor 1254, a predominant mixture of PCBs, altered nerve cell growth.
Our approach utilized a novel marine invertebrate embryo model designed to quantitate subtle mechanistic changes in early neuronal development.12 The surf clam, Spisula solidissima, was specifically chosen for the unique synchronisity of embryonic development within this species.4 Furthermore, neuronal growth in the surf clam is rapid and quantifiable during early embryonic development. To evaluate neuronal development, we chose serotonin as a biomarker of early nerve cell growth. 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.13 Involvement in nervous system development, learning behaviors, and memory6,14 makes serotonin a potential target of nervous system disruption by PCBs. Therefore, we specifically investigated whether or not Aroclor 1254 impairs serotonergic nerve cell development during embryogenesis.
Briefly, adult surf clams were collected by the Marine Biological Laboratory from Menemsha Bay, Martha's Vineyard, MA. In vitro fertilization was performed following the methods of Allen and Clotteau with moderate modifications.1,2 Fertilized oocytes were exposed to environmentally relevant doses of Aroclor 1254, ranging from 0 to 500 ppm, immediately following germinal vesicle breakdown to study the effects of post-fertilization exposure. Embryos were washed at 5 hours post-fertilization, transferred to 4 liter tanks at 6 hours post-fertilization, and maintained at 18°C in artificial sea water (30 ppt, pH 8.0). Samples of embryos were taken at 24, 48, and 72 hours post-fertilization and immediately fixed in 4% formaldehyde. Immunofluorescence techniques were used to identify serotonergic nerve cells as follows. Each embryo sample was divided into two groups and incubated in either serotonin antiserum or normal rabbit serum (control). All groups were subsequently incubated with a secondary antibody, goat anti-rabbit immunoglobulin labeled with the Oregon Green fluorophore. Confocal microscopy was performed on PCB-exposed and unexposed embryos to quantitate serotonergic nerve cell bodies. Following serotonergic cell body quantitation and image acquisition of individual embryos, sequential 1 micron sections were captured and three-dimensional analyses were used to reconstruct and rotate embryos to confirm the number of serotonergic nerve cells counted. Serotonergic nerve cell numbers were statistically analyzed with chi-square testing and regression analyses.
Our results demonstrated that embryos exposed to 1, 10, 100 and 500 ppm Aroclor 1254 showed a dose-dependent decrease in serotonergic nerve cell number when compared to the artificial sea water (0 ppm) and vehicle control embryos. This dose-dependent decrease was detectable as early as 24 hours post-fertilization, and was also seen at 48 and 72 hours post-fertilization. Chi-square analyses confirmed significant relationships between increasing Aroclor 1254 concentrations and decreasing numbers of serotonergic nerve cell bodies (p < 0.002, 0.003, and 0.002 respectively for 24, 48, and 72 hours). Regression analysis confirmed this relationship by showing a highly significant reduction of approximately 0.1 neurons per log unit of Aroclor 1254 concentration at each time point (p< 0.001 for 24, 48, and 72 hours). Therefore, these experiments showed that exposure to Aroclor 1254 significantly decreased the number of serotonergic nerve cells in developing surf clam embryos.
Disruption of the serotonergic nervous system during embryonic development may explain the correlation that has been documented between in utero PCB exposure and intellectual impairment. 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.7 Furthermore, serotonergic alteration may have profound effects on leaming and memory. A recent study by Mazer et al.8 reported that experimental depletion of serotonin during synaptogenesis led to neurodevelopmental disorders, including long-term alterations in neuronal morphology and function and an interference with spatial learning and memory systems. Therefore, PCB-induced disruption of the serotonin pathway during nervous system development may lead to permanent intellectual impairment. Further research must be conducted in order to assess the impact that neuronal impairment may have on aquatic animals, particularly those that rely heavily on nervous system function for essential survival tactics.
Acknowledgments
This project was supported in part by National Institute of Health grants T35DK07635 and RO1-CA44307, and by grants from the Geraldine R. Dodge Foundation and the Sweet Water Trust. The presenting author would like to thank IAAAM for a Student Travel Award, and WALTHAMTM for their support. In addition, the authors would like to thank Will Rand, Mark Martindale, Joan King, Eric Overstrom, Roxanna Smolowitz, Robert Palazzo, and Robert Brown for their contributions to this project.
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