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A model fish for pollutant studies

Mark Greeley admits the one-inch-long, brownish-gray zebrafish is a "rather drab aquarium fish." But he says the small striped fish "can be considered the mouse of the fish world, because it is arguably the best vertebrate model for development." Greeley then explains how to make a zebrafish look interesting: Turn it green.

Greeley, director of ORNL's Aquatic Toxicology Laboratory in the Environmental Sciences Division, is an ecotoxicologist who studies the effects of contamination on fish and relates the effects back to humans and to other wildlife, as well. He is particularly interested in endocrine-disrupting chemicals, artificial and natural substances that mimic or block the normal actions of hormones such as estrogen and testosterone. Endocrine-disrupting chemicals, including such common or well-known substances as organochlorine pesticides, plasticizers, dioxins, and PCBs, can cause significant health problems in wildlife and humans.

The risks of endocrine-disrupting chemicals are most apparent in fish and wildlife. "Downstream of sewage treatment plants, fish often have combined male and female sex organs from exposure to estrogenic compounds in pharmaceutical waste and other endocrine-disrupting chemicals," Greeley says. "Downstream of a paper and pulp mill, we have studied a fish population that consists entirely of males because of exposure to natural plant sterols and other hormone mimics in the wood-processing by-products. Endocrine-disrupting chemicals have been implicated in skewed sex ratios in birds, developmental abnormalities in frogs and other amphibians, deformed sex organs and other reproductive problems in alligators, and decreased fertility in a variety of wildlife species."

Endocrine-disrupting chemicals may also have subtle but no less significant health effects on humans. Although the evidence is less conclusive, these chemicals have been implicated in the development of human breast and testicular cancers, early onset of puberty in both boys and girls, lowered sperm counts, and decreased fertility in the developed world.

Greeley is using the zebrafish as a model to study the molecular toxicology of endocrine-disrupting chemicals. "The ultimate goal of this research," he says, "is to understand how these toxicants cause their adverse effects by discovering which genes are turned on and off and which proteins are produced or altered as a result of exposure."

To examine the behavior of specific genes as a result of exposure to endocrine-disrupting chemicals, Greeley and his associates—Suzanne Garnmeister, Kitty Mc-Cracken, and Zamin Yang—use a jellyfish gene that codes for a green fluorescent protein (GFP). The bioreporter GFP gene is first genetically fused to a hormone-responsive gene, such as the vitellogenin gene, which regulates the formation of yolk proteins in zebrafish and other non-mammalian vertebrate organisms. The resulting gene construct is then microinjected into fertilized single-cell eggs and incorporated into the zebrafish genome. Whenever the native hormone-responsive gene is expressed in these transgenic fish in response to natural hormones or from exposure to endocrine-disrupting chemicals, the bioreporter gene construct also turns on and glows with the typical intense blue-green fluorescent signature of the GFP gene. Because of the remarkable clarity of the zebrafish embryo, the researchers can determine exactly when and where the specific gene of interest is activated or turned off in response to toxicant exposure.

The simultaneous expression of potentially thousands of zebrafish genes during exposure to endocrine-disrupting chemicals is also being examined using a zebrafish DNA "tox-chip" microarray currently being developed in collaboration with Mitch Doktycz and Peter Hoyt of the Life Sciences Division (see Gene Chip Engineers). "Results with an early version of this chip," Greeley says, "clearly demonstrate the enormous potential of this tool in toxicological research."

Genes alone can't tell the whole story. Proteins produced as a result of gene expression actually mediate the toxic response.

"In collaboration with Brian Bradley of the University of Maryland–Baltimore County, we have demonstrated significant changes in protein expression in developing zebrafish following exposure to estrogen and estrogen-mimicking chemicals," Greeley says. "Proteins normally produced during early development were absent, but atypical proteins were produced. We expect the zebrafish to be an excellent model for studying the functional relationship between gene and protein expression in response to toxicant exposure.

"Because humans have many of the same genes, what we learn about gene and protein expression in zebrafish exposed to endocrine-disrupting chemicals and other toxicants will help us better understand what these chemicals can do to humans."

Zebrafish are becoming more fascinating now that they are giving scientists the green light to learn more about how toxins affect our genes.



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