Monday, March 16, 1998 ; Page A03

Ever since a group of schoolchildren discovered grossly deformed frogs in a pond near Henderson, Minn., in the summer of 1995, scientists around the country have been struggling to understand what may be causing the abnormalities, which have subsequently been discovered in a number of other states.

Now, a low-budget, carefully targeted research effort appears to have produced a significant finding in the long-running scientific mystery. Later this week, a group of researchers led by David Gardiner of the University of California, Irvine, and Bruce Blumberg of the Salk Institute in La Jolla will report new evidence linking the frog deformities with exposure to substances known as "retinoids."

Retinoids, compounds that are derived from Vitamin A, include the powerful hormone retinoic acid, which regulates several key aspects of development in all vertebrates, including humans. Exposure to excess amounts of retinoic acid is known to produce birth defects. In humans, for example, the retinoid-based acne treatment Accutane has produced birth defects when used by pregnant women.

Taking different approaches, Blumberg and Gardiner discovered retinoids in water samples from a Minnesota lake that has produced many deformed frogs, plus evidence that the limb abnormalities in frogs from the site were in fact caused at least in part by retinoids.

The researchers stress that the findings, which will be presented at the Midwest Declining Amphibians Conference in Milwaukee, are preliminary and point only to the need for further work, not to a final answer. But the results are significant, they said, because two independent lines of inquiry implicate retinoids, and because of the human health risks of retinoid exposure.

"Bioactive retinoids in water are a definite public health risk," said Blumberg. "Retinoids cause developmental deformities in every vertebrate species that's been tested, from primitive fish to humans."

Frogs with extra legs, missing legs or leg parts, bizarre skin webbings, missing eyes and a variety of misshapen legs have been found throughout Minnesota, as well as in several other states, including Vermont, Oregon and Delaware, prompting investigations by a number of state and federal agencies. Frogs with similar deformities have been under study in Quebec by the Canadian Wildlife Service since 1992.

Scientists are concerned about the frog deformities, as well as the possibility that amphibians in general may be declining around the world, because some biologists consider amphibians "sentinel species" that can signal serious environmental problems early.

A variety of possible explanations have been proposed for the frog deformities, ranging from relatively innocuous natural causes to possibly more alarming toxics, such as pesticide pollution.

In September, the Minnesota Pollution Control Agency (MPCA) and the National Institute of Environmental Health Sciences (NIEHS) announced that water from private wells in Minnesota had produced deformities in laboratory frogs, and began distributing bottled water to people whose wells were near sites with deformed frogs.

The announcement was criticized by other scientists who objected that the test procedure was flawed and the drinking water warnings premature. The MPCA and NIEHS now acknowledge that further tests show no evidence of contamination in those wells -- or at more than two dozen others added to the study since then. But the picture remains muddy because five additional wells tested recently do appear contaminated and not all of them are near sites with deformed frogs. Together, the MPCA and NIEHS have spent nearly $1 million on the frog problem.

Gardiner and Blumberg said their research, which so far has cost $5,000 plus some lab time and materials, began last fall, after a two-day brainstorming session with colleagues who felt the larger investigation had failed to aggressively pursue the retinoid scenario. The meeting was organized by Gardiner and his wife, Susan Bryant, a prominent limb development expert at the University of California, Irvine.

"Retinoids are clearly the place to start," said Gardiner. "This is a problem in development and developmental biologists have the resources available to attack the problem."

Gardiner contacted David Hoppe of the University of Minnesota, Morris, the state's leading field investigator, and obtained 29 frog specimens. In November, Gardiner went to Minnesota and collected water from the lake where the frogs were found.

Blumberg tested the water with a sensitive assay that measures the activation of human retinoic acid "receptor" proteins. Those receptors regulate genes in human cells that are critical in limb development and patterning when they are switched on by retinoic acid. The water tested positive.

Meanwhile, Gardiner and Bryant examined the frogs with a commonly used procedure for clearing the animals' soft tissue while staining the cartilage and bone. The result is a transparent "visible frog" in which the skeleton is dyed a deep blue.

In every deformed frog examined, one or more leg segments seemed to be growing back on itself in reverse, producing a "triangulated" appearance. These unique "bony triangles" have turned up repeatedly in past retinoic acid experiments. Gardiner and Bryant eventually concluded that bony triangles are a signature of retinoid exposure.

"We thought we'd never seen anything like that before," said Gardiner. "But when we looked back at the literature, there they were. In chickens. In mice. In several species of frogs."

The retinoid, or retinoid-like substance, detected in the Minnesota water could be a pesticide or a derivative of one, Blumberg said. It is also possible that it's a natural compound produced by microorganisms or plants in the lake.

"If it's natural in origin that just means there's nobody to blame," he said.

Gardiner and Blumberg think there may be more than just a retinoid behind the frog deformities. While it is certain that a retinoid could cause abnormal leg development, evidence that retinoids alone can induce entire extra legs is mixed.

Jim Burkhart, a research biologist who heads the NIEHS frog investigation, said his agency recently found retinoids in water samples from several sites in Minnesota using a test slightly different from the one developed by Blumberg. The Environmental Protection Agency, which last year tested the insecticide methoprene for possible retinoid properties, is also now preparing a more general retinoid assay it will begin using this spring.

By then, Gardiner and Blumberg plan to be working on more water samples. But they also hope to do something that no one else has yet managed. "I think what we've got to do now is show people that one last piece of the puzzle," Gardiner said. "We're going to have to produce a frog with extra legs in the lab."

Cutline: A deformed juvenile mink frog (Rana septentrionalis) collected in Minnesota. This one has several extra legs protruding from its sides.

THE DEFORMED FROG MYSTERY (This graphic was not available)

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