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A scientist at the Marine Biological Laboratory (MBL) has published the results of an EPA-funded clam embryo study that supports her hypothesis that, when combined, the pollutants bromoform, chloroform, and tetrachloroethylene--a chemical cocktail known as BCE--can act synergistically to alter a key regulator in nerve cell development. While scientists have previously studied the effects of these pollutants individually, this is the first time anyone has demonstrated that BCE's components can work together to adversely affect neuron growth in a model organism. The study, which is reported in the January 2005 issue of Environmental Toxicology and Pharmacology, is the first step toward understanding how exposure to BCE might affect human nerve cell development--knowledge that may one day provide clues about such neurological mysteries as autism spectrum disorder or attention deficit hyperactivity disorder.

To test her hypothesis, MBL scientist Carol Reinisch and her colleagues treated developing surf clam embryos (Spisula solidissima) with different combinations of BCE and studied their effects on nerve cell growth. "On a cellular level, clam neurons are extremely useful in studying basic mechanisms of cell development," says Reinisch, an expert in PCB-induced neurotoxicity. "Of the different combinations and strengths of BCE components tested, we found that all three together induce the greatest adverse response. Treating the embryos with the triple mixture resulted in increased production of a subunit of an enzyme called protein kinase A (PKA), which previous research suggests plays a role in neural development," says Reinisch. "Fluctuations in PKA may influence not only neuronal maturation but also how neuronal networks are constructed during development," she says. Alterations of this enzyme may affect neural development and neural connections by activating or inactivating other proteins. Demonstrating that clam embryos are affected by BCE paves the way for additional studies that may help explain how exposure to BCE affects human nerve cell development and how it might relate to neurological disorders. "We can clearly state that we found an increase in a component of PKA, and PKA is known to be involved in neural development. The BCE mixture is capable of altering neural development, and alterations in neural development are thought to be a cause of autism," says Jill Kreiling, first author of the paper and a member of Reinisch's lab. But Kreiling cautions, "We cannot say at this time if the alteration we see in clam embryos is the same alteration that causes autism. That will require future research." In fact, Reinisch and Kreiling have already begun the next phase of their work. They are currently examining the origins of BCE toxicity at the single-neuron level to learn what genes are turned on and off by the exposure to the chemical mixture. The research is focused on a family of genes known as P53, which helps to regulate the cell cycle. They have also moved their studies to a new model system: the zebrafish, a vertebrate with more similarities to humans. Reinisch's work on BCE is funded by a STAR grant from the United States Environmental Protection Agency. Science supported by the STAR program is rigorously peer reviewed. Study Background and Research Methods When MBL researcher Carol Reinisch first heard news stories about Brick Township, New Jersey--a town that was the center of an ATSDR* study in the year 2000 that documented a higher than usual rate of autism spectrum disorder and explored its link to bromoform, chloroform, and tetrachloroethylene in Brick's water supply--it got her thinking. "My hypothesis was this: While it had been proven that individually the chemicals don't impact nerve cell development, the combination of all three might pose increased risks," she says. So Reinisch decided to test the chemical cocktail, in similar proportions to those found in Brick, on a species of surf clam readily available in the waters off of her Cape Cod lab. The species is famous among scientists as a model for the study of cellular processes that are common among all animals, including humans. Reinisch and her colleagues collected surf clam eggs and sperm, fertilized them in vitro, and added BCE to normally developing clam embryos. The investigators used a combination of antibodies and dyes to show the presence of neurotransmitters in single neurons. "We've actually taken superlative three-dimensional pictures where we go right through the embryo," says Reinisch. The sequence of images yields a three-dimensional representation of the embryos' developing neurons. Reinisch and company examined periodically recorded images to see what effect, if any, the chemicals had. They also measured the levels of proteins in the normal and treated embryos to check the visual data. Read the full paper


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