Answers Near in Deformed Frog Mystery

In a cluttered UC Irvine science laboratory about 2,000 miles from the bucolic lake where the alarming amphibians were first spotted, the mystery of the deformed Minnesota frogs may be close to being solved.

It was here amid the microscopes and test tubes that David Gardiner and Susan Bryant, husband and wife biologists, identified their “prime suspect” in the now-famous case of the six-legged frogs--or nine-legged or legless or any of the many other varieties of bizarre deformities that have since been found in frogs in several states.

And it is here, in the university’s “leg lab,” that little tadpoles are growing, swimming about in tiny petri dishes and getting ready to metamorphose into frogs. When they do, Gardiner and Bryant will try to duplicate in the lab what some Minnesota schoolchildren found in the wild more than two years ago.

The suspected culprit in the deformities, Gardiner announced last month to a conference of scientists in Milwaukee, is retinoids, hormone-like substances that are essential for limb growth but can cause major deformities when an organism is exposed to excess amounts.

Derived from vitamin A, retinoids are found in humans, frogs and other vertebrates naturally, and are produced by lake plants and other natural organisms. They also can come from pesticides, Gardiner and Bryant said.

It is much too early to point fingers at the source, Gardiner said. More experiments are now underway by their partner Bruce Blumberg at the Salk Institute in La Jolla to definitively determine whether retinoids are at work. The experiments will make clear whether chemicals in the lake water are acting like retinoids and binding to retinoic acid receptors, which activate the developmental pathways for limb growth. Gardiner said they expect to have an answer by summer.

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Identifying the culprit is crucial, Gardiner and Bryant say, and not just for the frogs. This is about humans too, for our species grows legs using the same developmental paths as frogs, they point out. And if something is making the frogs grow deformed, extra limbs or no limbs at all, they ask, what can it be doing to us humans?

The mystery is being unraveled in Southern California “because nobody knows weird legs like we do,” said Bryant, a biology professor.

She and Gardiner, a UC Irvine researcher, have spent years exploring how limbs can be regenerated, finding the secrets to normal growth by examining abnormal growth.

Gardiner, Bryant and Blumberg are not alone in the search for an answer to the frog puzzle. Both the federal Environmental Protection Agency and the National Institutes of Health have sponsored numerous studies--testing the waters, studying the effects of ultraviolet rays, looking for anything that may be causing the frogs’ deformities.

“It’s incredible all the stuff they are doing, but you know what? Nobody looked at the frogs,” said Gardiner, whom the EPA invited to share his perspective as a developmental biologist at a workshop on the frogs a year ago, months before he developed the retinoid theory.

“So we looked at it and gave them the answer they didn’t want to hear, which was ‘You’ve got a serious problem here,’ ” Gardiner said. “I came back and thought that they were going to call any day to get us started working on it.”

After waiting futilely for the call, Bryant and Gardiner took the initiative, organizing a workshop of developmental biologists. The consensus was “Yes, these animals are being affected during embryonic development . . . and that whatever it was that was affecting the developmental pathways here could have an effect on humans as well,” Gardiner said.

He and Bryant then contacted Minnesota’s chief investigator on the frog mystery, and Gardiner went to collect frogs from the lake.

Using a stain that makes soft tissues transparent and bony tissues stand out, similar to an X-ray, Gardiner and Bryant examined the frogs’ legs for clues.

After staring for hours at countless legs of all shapes and sizes, suddenly they saw a common malformation: The bones, in one frog after another, formed triangular patterns. Instead of growing straight, the bones doubled back in the middle, so that the two ends were near each other and the middle was bent, like an elbow.

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They knew from their own work and from published studies that a bony triangle is the signature of retinoid exposure, Gardiner said. In research, retinoic acid can be used to purposely disrupt limb development, so that scientists can better understand normal growth, he said. Retinoids are known to cause deformities in human embryos, the couple said. For example, pregnant women are told not to use retinoid-based skin creams.

“We don’t know how retinoids do it [make bony triangles], but we haven’t found anything else that does this,” Gardiner said.

Retinoids, he said, “are sensitive. If you do anything to disrupt the normal metabolism of retinoids, then you will completely screw everything up, they are so powerful.”

If Blumberg’s assays of the lake water reveal that retinoids are indeed present, the path for further research will be clear-cut: using retinoids to duplicate the deformities in frogs at the lab, which would help scientists search for the source at the lakes. If the experiments in La Jolla find no retinoid-like substances, the course of further work is more difficult, Gardiner said.

While federal agencies are “spending millions of dollars” on a “shotgun approach” to solving the frog mystery, the work of Gardiner, Bryant and Blumberg has cost just a few thousand dollars, scratched together from a few sources, Gardiner said. Not that the federal agencies aren’t interested in his work. Gardiner stays in touch with the National Institute of Environmental Health Sciences, part of the National Institutes of Health, and has been contacted by the EPA.

Although the EPA is focusing its investigation on ultraviolet rays, Gardiner believes that the culprit is clearly in the water. Species of frogs whose eggs and larvae spend little time in the water are less affected than those whose eggs and larvae spend long periods developing in water, he said.

The deformed frogs have garnered publicity nationwide, but reaction varies, Gardiner said.

“People in Minnesota want to know: ‘What should I do?’ ” he said. “They’re worried about their kids, about themselves.

“Then you talk to somebody in Southern California and they go, ‘Wow, sounds like a real problem for frogs.’ ”

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Gardiner said the implications of the frog deformities did not fully hit him until he was flying home from Minnesota last year, after collecting frogs and having lunch with a family that lives at the lake.

He was reflecting on the argument posed by some scientists that the culprit cannot be pesticides because those chemicals are used everywhere, while deformed frogs are showing up only at certain sites.

“I thought, ‘Wait a minute. Another way to think about it is, if it is a pesticide, and it is widely used, and if it has caused deformed frogs in Minnesota, what the hell is it doing in Costa Mesa and Newport Beach and Irvine?’ ” Maybe frogs in Southern California have not been affected because this area does not have the same climate or lakes, but perhaps the chemicals have different effects on different terrain, he said.

“That’s when all of a sudden I realized this had nothing to do with living in Minnesota,” Gardiner said. “Now I’m worried about it, you know? I’m very worried about it.”