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fish development or affect fish diseases. “We’re seeing a growing interest in zebrafish for high-throughput toxicity testing,” he says. Data on the use of laboratory animals are not collected in the U.S. But in the U.K., which does track animal use, the number of experiments that used fish increased 103% from 1996 to 2006, compared with an 8% increase in the use of mice, rats, and other rodents over the same period, according to government data.
A Tale of Two Fish REBECCA RENNER
ROBERT TANGUAY
SHUTTERSTOCK
From rapid screens to detailed studies, scientists are reeling in more fish.
Transgenic zebrafish that express green fluorescent protein in spinal motor neurons allow scientists to study spinal neurons in the live animal. By now it is a cliche´ of environmental sciencesthere are too many chemicals in use commercially and not enough resources to test for their potential toxicity. In the U.S., approximately one-quarter of the 70,000 chemicals in use have never been tested for toxicity, and every year the problem grows as new chemicals come onto the market. “We are not serving the public well in terms of evaluating whether new drugs or chemicals pose a significant risk to humans,” says developmental toxicologist Robert Tanguay, who directs the Sinnhuber Aquatic Research Laboratory at Oregon State University. “The test requirements are so expensive that they are often not done.” To cope with this problem a 2007 National Research Council report, Toxicity Testing in the 21st Century: A Vision and a Strategy, called for less reliance on animal studies and more on in vitro tests with human cells. That is the goal, Tanguay says. “We want to find a molecular signal, the ultimate precursor to an adverse effect. But that remains a long way off, because we currently can’t even predict what an individual cell will do in response to exposures, let alone predict how all the different cells in an organism will interact following a chemical exposure.”
The Big Screen In the meantime, Tanguay and a growing number of researchers are tackling chemical screening by using small fish, in particular zebrafish, and adapting the high-speed methods that are used for in vitro testing. The demand for such testing is growing rapidly, according to Randall Peterson, an assistant professor of medicine at Harvard Medical School. As a window into the mechanisms that affect humans, Peterson uses zebrafish and highthroughput chemical screens to find chemicals that perturb 6784
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The small fish that are popular with toxicologists include medaka, killifish, guppies, and for ecotoxicology research, fathead minnows. But the field is dominated by zebrafish (Danio rerio), which is a familiar denizen of home freshwater aquariums. The 2-inch-long striped speedster from the Ganges River has been used to study development for more than 25 years.
See-Through Embryos Zebrafish have many characteristics that endear them to researchers. They are fecund. Each week, females lay 200-300 eggs. They also have see-through embryos that grow rapidly outside the mother’s body. Just 4 days after fertilization, the young zebrafish can see and swim; they have well-developed hearts but primitive nervous systems. And yet genetically, zebrafish have much in common with humans. Among other findings, detailed zebrafish studies have provided insights into the workings of the heart, blood clotting, and degeneration of the retina. The wide array of tools available to researchers include knockout fish, in which genes are suppressed, and knockdown fish, in which the production of a particular protein is inhibited. In 2004, the EU awarded 12 million euros to the ZF-MODELS consortium to study zebrafish models for human development and disease. ZFIN, an information source for the zebrafish research community, has more than 3000 members. For high-throughput assays, Tanguay uses a container with 384 small depressions or wells and puts about four zebrafish embryos in each well. “We know that development happens precisely,” he says. “So, for a simple screen, we can use a simple endpoint that can be seen at the microscopic level.” This endpoint might be a change in behavior or a physical trait such as a bent spinal cord, a heart defect, or 10.1021/es801813m
2008 American Chemical Society
Published on Web 08/27/2008
GAYLE ORNER, COURTESY OF ABBY BENNINGHOFF
researchers think that too much activity in these peroxisomes can increase oxidative stress, produce DNA damage, and ultimately lead to cancer. But peroxisome proliferation is much less of an issue with humanssor troutspossibly because humans and trout have fewer receptors that turn on the process of peroxisome proliferation.
ROBERT TANGUAY
the development of just one eye. “We can screen thousands of chemicals a day,” he says. The zebrafish screen is one of only a few that use a living animal among the multitude of screens in the U.S. EPA’s ToxCast program, which aims to develop cost-effective ways to screen large numbers of chemicals. Most of the others are in vitro screens with single cells or proteins. “Since the zebrafish screen is developmental, we hope that it will look at the ability to cause birth defects,” says Robert Kavlock, director of EPA’s National Center for Computational Toxicology.
Rainbow trout studies can shed new light on cancer mechanisms.
Zebrafish embryos exposed to the soil fumigant sodium metam develop distorted or twisted notochords compared with controls. Regulatory requirements, however, lag behind researchers’ enthusiasm. According to Laurence Musset, principal administrator at the Organisation for Economic Co-operation and Development (OECD) Environment Directorate, no OECD test guidelines exist for fish-testing methods that are aimed at assessing human health, and there is no proposal for such a test guideline. “Certain fish tests,” she notes, “in particular for screening endocrine disrupters, can provide indication of mechanisms of action, but whether these mechanisms of action can be extrapolated to humans is not certain.”
Trout Fishing for Cancer Although zebrafish are comparative newcomers to toxicology, the use of trout to study cancer is long-standing. It started in the 1960s after an outbreak of liver cancer in hatchery trout. The culprit turned out to be aflatoxin, a potent carcinogen found on moldy peanuts and grains. Recently, toxicologist Abby Benninghoff and her colleagues at Oregon State University used aflatoxin and rainbow trout to shed light on the puzzle of perfluorooctanoic acid (PFOA) and liver cancer. The source of PFOA’s ubiquitous presence in the blood of people from developed countries is a mystery, as is the toxicological significance of this finding. Studies have shown that relatively high levels of PFOA can cause liver cancer in rats. But the relevance of these studies to humans is in doubt because PFOA is a potent peroxisome proliferatorsit causes a proliferation of these microbodies, which are involved in fat metabolism, in the liver. Many chemicals have this effect; among them are clofibrate and similar hypolipidemic drugs, phthalates, and organic solvents such as trichloroethylene. Although the details of why peroxisome proliferation causes liver cancer in rats and mice are not known,
For PFOA, the evidence is mixed: mice with the genes for these receptors knocked out still get enlarged livers when fed PFOA, suggesting that something else is going on. In addition, PFOA induces testicular tumors in rats, and this effect is correlated with elevated levels of serum estradiol. So Benninghoff and colleagues wanted to see whether such a mechanism might be associated with liver tumors. “Laboratory rodents make very good animal models for many cancer studies, but this may not be one of them. Our data indicate that the book on PFOA and liver cancer is not closed,” she says. To tease out the way in which PFOA is linked to liver cancer, Benninghoff started with 1000 juvenile trout. First, she exposed the trout to aflatoxin to initiate the formation of tumors, and then she fed them different peroxisome proliferators to see whether the chemicals would promote tumors. Some trout ate PFOA; others ingested clofibrate or dehydroepiandrosterone (DHEA), a natural hormone precursor that is a peroxisome proliferator in rats but not in trout. Between 10% and 30% of the fish exposed to aflatoxin developed at least one liver tumor. But 60-70% of the animals that ate PFOA as well developed liver tumors, sometimes six or more. DHEA also enhanced the incidence and number of tumors, but clofibrate did not. Among the tools used to examine the possible mechanism of action were enzyme activity assays, levels of a biomarker protein in the blood, and gene expression profiling. PFOA and DHEA showed evidence of estrogenicity, whereas clofibrate did not. “The data from these different assays work together to support our basic finding that, in trout, PFOA does not act as a classic peroxisome-proliferating chemical but rather promotes liver cancer via an estrogen-like mechanism,” says Benninghoff. She also cautions that the amount of PFOA given to the animals was highshigher than typical human exposure. “The goal was to determine if it was possible that PFOA promoted cancer via an estrogen-like mechanism,” she says. “To get to the question of risk of exposure for humans, we would need to conduct additional studies using PFOA at concentrations that are found in people,” she adds. At one time, toxicologists believed that studies conducted on rats, mice, and other animals that are phylogenetically close to humans yielded experimental data more relevant to humans. But as studies with zebrafish and rainbow trout show, when toxicologists go fishing for results, they can gain a detailed mechanistic understanding and save time and money, too. ES801813M VOL. 42, NO. 18, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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