POLYCHLORINATED BIPHENYLS ARE SELECTIVELY NEUROTOXIC IN THE DEVELOPINGSPISULA SOLIDISSIMAEMBRYO

Abstract

Polychlorinated biphenyls (PCBs) are ubiquitous environmental pollutants that accumulate to toxic levels in the food chain. Using Spisula solidissima (surf clam) embryos as a developmental model, it was shown that Aroclor 1254 specifically targets two neuronal structures during embryonic development. Embryos were exposed to 1, 10), or 100 ppm Aroclor 1254 or an acetone vehicle control posthatching for 24, 48, and 72 h. Embryos labeled with a serotonin antibody or a neural antigen antibody and a rhodamine-conjugated secondary antibody were viewed by confocal microscopy. The cerebropleural ganglion showed a decrease both in serotonin production and in the size of the serotonin-synthesizing region upon exposure to 10 and 100 ppm Aroclor 1254. These decreases were detectable as early as 48 h postfertilization. When exposed to 100 ppm Aroclor 1254, the primitive neural plexus, which coordinates the movements of the mouth and velum, showed a delay in onset and cessation of expression of a molluscan-specific neural antigen. Exposure to Aroclor 1254 did not affect the overall growth and morphology of the embryos. In addition, analyses of total protein profiles and heat-shock protein 70 levels showed that exposure to Aroclor 1254 did not trigger protein degradation or cause a stress or shock response. These results show that exposure of Spisula embryos to Aroclor 1254 specifically targets neurogenesis while having no effect on the overall growth of the embryo.

A new invertebrate member of the p53 gene family is developmentally expressed and responds to polychlorinated biphenyls.

“…Thus surf clam p53 family members are not only differentially expressed during normal development, but are also differentially responsive following exposure to an environmental stressor. In addition, the reduction in p120 at 48 hr and beyond corresponds with the phenotypic expression of PCB-induced toxicity in surf clams, a reduced serotonergic nervous system (17).…”

“…At the swimming blastula stage, embryos were transferred to 4-L aerated tanks at a concentration of 100 mL. For the p53 protein analyses, larvae were cultured at a density of 1,000 mL in stirred, 250-mL containers as described in Kreiling et al (17). Neither culture method caused significant stress or mortality.…”

“…In both cases, nonspecific binding was blocked with 5% nonfat dry milk. Immunoreactivity and sample quantification for Western blots probed with either antibody were determined as described in Kreiling et al (17). The signals from four to five separate embryo cultures were integrated across each time point or treatment and normalized to the maximum signal in that culture as a percentage of the maximum (set at 100).…”

“…Spisula embryo samples were treated as described in Kreiling et al (17), transferred to 8% Laemmli gels normalized to 2 or 4 µg total protein per lane (unless otherwise cited), and transferred with Dunn carbonate/bicarbonate/methanol buffer onto nitrocellulose (27). One set of blots was probed with a goat polyclonal antibody (1:500 or 1:1,000 dilutions) made by New England Peptide (Fitchburg, MA) to a 24-amino acid peptide sequence generated from surf clam p120.…”

“…PCBs are lipophilic, environmentally pervasive chemicals that accumulate in plant, animal, and human tissues, promote tumor formation, can damage DNA (14,15), and induce neurotoxic effects during early development in clams (16,17) and humans (18). In vitro fertilization and subsequent developmental studies are easily conducted in the surf clam, Spisula solidissima (16,17,(19)(20)(21), but to our knowledge are not successful in either soft-shell clam or squid. During investigation of these embryos we partially cloned, sequenced, and generated a multiple alignment for a new member of the p53 gene family that codes for an exclusively embryonic 120-kD protein.…”