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