| June 13, 2002
Zebrafish: Drug Discovery of A Different
Stripe Holds Promise In COX Research
PA) -Researchers at the University of Pennsylvania
School of Medicine have discovered that zebrafish
produce enzymes analogous to the two human cyclooxygenase
(COX) enzymes - COX-1 and -2 - proteins that have a
role in a variety of ailments including cardiovascular
disease, some types of cancer, and arthritis. Furthermore,
drugs that target the COX enzymes, nonsteroidal anti-inflammatory
drugs (NSAIDs) and selective inhibitors of COX-2, seem
to act in a manner similar to humans.
"We have learned a great deal about how the COX
enzymes and their inhibitors work from mouse models
of COX gene inactivation," said Garret A. FitzGerald,
MD, Chair of the Department of Pharmacology and
director of the Center for Experimental Therapeutics
at Penn. " However, these systems have their limitations.
The zebrafish promises to play a complementary role
in which both biology and the role of drugs can be investigated."
Traditionally zebrafish have been useful in searching
for gene mutations induced by exposure to toxic chemicals,
which coincide with abnormalities of development. Such
an approach is termed "forward genetics."
The Penn paper, detailed in this week's print edition
of the Proceedings of the National Academy of Sciences,
is an early example of the use of zebrafish for "reverse
genetics." A particular set of genes - in this
case, the COXs - are sought out and their function
is uncovered by manipulating their action in zebrafish.
The COX enzymes produce prostaglandins, fatty acids
that perform a number of hormone-like tasks.
Prostaglandins alter the activities of the cells near
and around where they are made. They also cause inflammation
and can regulate blood flow to some organs, transport
across cell membranes, and transmissions between neurons.
The COX proteins are encoded by separate genes on separate
chromosomes and, likewise, have two separate but interdependent
functions. COX-1 is responsible for the "day-to-day"
production of prostaglandins, while COX-2 is highly
regulated by numerous other cellular signals when needed.
Drugs which selectively target COX-2, such as Pharmacia/Pfizer's
Celebrexâ and Bextraâ and Merck's Vioxxâ
have attracted much attention. They have been remarkably
successful, based on claims that they relieve pain and
inflammation while lessening the risk of stomach ulceration
and bleeding associated with traditional NSAIDs. One
issue of controversy has been whether the benefit of
COX - 2 inhibitors in the stomach is offset by an increased
risk of heart attack.
"The zebrafish has particular advantages for the
study of drug action," says Tilo Grosser, MD, Research
Associate in Pharmacology and first author of the study.
"The embryos are translucent, so we can study the
pattern of gene expression during development, as well
as in the adult. The near completion of the zebrafish
genome project allows us to hunt for relatives of human
genes of interest. Then we can manipulate them and see
how they function."
Indeed, this is just the approach that Grosser, FitzGerald
and their colleagues took in the present study. They
thought the zebrafish might be a particularly useful
model of the cardiovascular effects of COX inhibitors.
First, they identified the zebrafish versions of the
COX genes and showed that they behaved like the human
enzymes. Next they studied their distribution. Interestingly,
both COX- 1 and COX-2 were extensively expressed in
the blood vessels of zebrafish, but with different patterns
In humans, prostaglandin products of COX-1 causes blood
vessels to constrict and platelets to become sticky
- the first step in a heart attack or stroke. COX-2
products, by contrast, are formed in blood vessels where
they dilate blood vessels and prevent the activation
of platelets. NSAIDs and aspirin block both forms of
the enzyme. COX-2 inhibitors leave the hazardous COX-1
products unaltered, thereby perhaps predisposing some
individuals to a risk of heart attack.
"We were impressed that COX inhibitors behaved
much the same in zebrafish as in humans," said
Grosser. An NSAID blocked the stickiness of zebrafish
thrombocytes -zebrafish platelets - and prolonged the
bleeding time, while a COX-2 inhibitor failed to exhibit
these cardioprotective properties.
The researchers also illustrated the value of this model
system by "knocking down" the COX genes. This
revealed a new role for COX-1 during development, one
that may have been masked in mice and humans by maternal
formation of prostaglandins.
The high fecundity of zebrafish makes them a particularly
attractive species for high thruput screening in the
selection of new drugs. "Rapid acquisition of 'proof
of principle ' is a particular challenge for the pharmaceutical
industry, " said FitzGerald. "It is our hope
that the zebrafish will facilitate this effort considerably."
This work was supported by the Alexander von Humboldt
Foundation and the National Institutes of Health.
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For an image of the COX-1 and COX-2 genes , click
Please credit the University of Pennsylvania School
of Medicine for the image.
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