Oct. 29, 2004 DURHAM, N.C. -- A primitive roundworm called Caenorhabditis elegans (C. elegans) is being evaluated in a Duke University laboratory as a cheaper and quicker alternative to rats and mice in testing chemicals for several kinds of toxicity.
In its natural environment, C. elegans spends its brief life dining on microbes in the soil. But Jonathan Freedman of Duke's Nicholas School of the Environment and Earth Sciences envisions that, in a laboratory setting, these simple animals could substantially reduce, and in some cases perhaps eventually replace, the need for expensive, large-scale rodent studies
The tiny roundworm has long been a favorite among molecular biologists and health researchers since its 959 cells contain many genes and proteins that function similarly to those of higher animals, including humans. Freedman, an associate professor of environmental toxicology who has worked with C. elegans since graduate school, now seeks to apply this extensive knowledge about the roundworm's biology to answer questions involving toxicology.
"The idea is to quickly screen chemicals with C. elegans so you don’t have to do so many mega-rat studies," Freedman said in an interview. "If Company X thinks it has a chemical that may be a nerve toxin or cause cancer, we will put it through our system to help find out. What we've done is save that company millions of dollars because it no longer has to do as large a rat study.
"It can cost a company $10 million and it may have to go through 100,000 rats over a year or two just to do a complete study on one chemical. With our worms, I envision we’ll be able to get the whole thing done in a couple of weeks to maybe a month."
Freedman has a $2.4 million three-year contract with the National Toxicology Program (NTP) to evaluate the feasibility of such a "high-throughput" C. elegans toxicity testing system that will use robotic equipment to mix chemicals and sort worms efficiently.
During this evaluation, his group plans to expose developing roundworms to 200 different chemicals, in collaboration with researchers at the NTP and Environmental Protection Agency. The group will also evaluate how chemicals affect a roundworm’s neural systems at various stages of life.
Freedman foresees several advantages if researchers use nematodes instead of the rats and mice that are now the laboratory standards for such work, especially during the expensive initial stages that typically involve large-scale screening.
For example, it takes just 3½ days for the roundworm to develop from egg to adult. Since each adult worm is only 1 millimeter in length, large numbers can be maintained and tested in small spaces.
Because the roundworms are transparent, researchers can also directly monitor chemicals effects on the worms' developing internal organs.
Toxicity screening during development evaluates how pre-selected amounts of chemicals affect groups of animals as they grow. Even if the animals do not sicken or die as they mature in the presence of toxicants, the chemicals may affect their organs in ways that can be investigated - normally through surgery or necropsy.
As with laboratory rats and mice, Freedman and his workers can even produce "knockout" varieties of C. elegans to evaluate how the animal's physiology changes if specific genes are excluded from its genome through biochemical manipulation.
Unlike genetically engineered rodents, the roundworms themselves are not engineered. Instead, their bacterial food is simply spiked with "antisense" DNA designed to block the function of the gene. "To knock out one mouse gene can cost $100,000 to do a genetics study that takes a year," Freedman said. "Whereas in C. elegans we just feed the roundworms a strain of bacterium and the gene is knocked out."
In addition, strains of the roundworms have been genetically engineered in Freedman's lab and others to make various cells change color or emit a fluorescent glow in the presence of a toxic chemical.
Freedman’s funding source, the NTP, coordinates toxicological testing programs within the U.S. Department of Health and Human Services and seeks to increase scientific knowledge about potentially hazardous substances. As part of a new initiative to broaden and refine its investigational methods, NTP is extensively evaluating C. elegans’ potential value to developmental toxicology.
"We know basically everything about the development of this particular organism through its entire lifespan," said NTP associate director Christopher Portier. "Since we're interested in finding something that, while it won't be definitive, can warn us about chemicals that could potentially affect development, this is an extremely good model for doing that."
Using standard rats and mice, "we can only do so many assays is a given year," he added. "It’s just a matter of resources, space and availability of animals. So the idea is to develop an inexpensive, fairly sensitive screen that can give us guidance on what to test."
"Potentially, if we get enough information put together and we feel comfortable enough with C. elegans, it may in fact replace the rodent. But for now what we're looking for is something that helps us set priorities on what to test in rodents."
As his multimillion dollar contract begins, Freedman has seen his Nicholas School lab triple in size to more than 15 people. Along with the new hires have come boxes of specialized equipment that give his part of the university some of the ambience of high-tech industry.
Much of the work is being done by computer-controlled robotic machines. Robots automatically dispense hot agar gel to support the bacterial colonies the worms use as food. After the agar cools and solidifies in the sunken wells where the animals will live, machines add drops of bacteria.
Robots also measure out chemicals at various levels of dilution and place those in the wells. Meanwhile, another machine, called a "biosorter," sends worms through a centrifuge before sucking them into tiny passages. There they are counted and sorted one by one while a laser beam senses each animal’s length, as well as its diameter, age, color and general health. Then the biosorter dispenses the right worms into the correct wells for a specific experiment.
There are 96 wells arrayed on each plate, the basic testing unit of this highly automated operation. Between 10 and 50 nematodes are placed in each well depending on the test. "We may put worms that have just hatched into a well and watch them grow," Freedman said. "Or we’ll do another experiment where we put an adult animal in and see how many offspring it makes and count the offspring and see how fast they grow.”
Another variation is to create varieties of transgenic worm lines, each bearing a different gene that fluoresces green under stress. "That way in a 96-well plate each well would have a different strain of transgenic worm that could respond to a chemical," he said. "So you just put the same chemical in all the wells to find out which gene is turning on."
Freedman's lab is also equipped to evaluate the activity of individual genes more directly. "We will look for changes in the expression of every gene in the animal caused by a given chemical," he said. "We need to come up with microarray fingerprints for each of the toxins." Whole flasks of worms can be grown and dosed with chemicals before having the messenger RNA extracted from their cells.
The amount of each messenger RNA molecule, which carries instructions from genes on how to build proteins, can then be measured on custom microarrays and compared with the genomes of nematodes that have not been exposed to chemicals.
"We will look for changes in the expression of every gene in the animal caused by a given chemical," Freedman said. "We need to come up with microarray fingerprints for each of the toxins."
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