June 15, 2011 (Vol. 31, No. 12)

Polliwogs Sport Metabolic and Immune Systems Closer to Human than other Vertebrates

Research at the Museum National d’Histoire Naturelle in Paris spawned the French biotechnology firm WatchFrog. Co-founders Gregory Lemkine, Ph.D., and Barbara Demeneix, Ph.D., met while working at the museum. Dr. Demeneix, a molecular physiologist and endocrinologist, created methods for following transcriptional regulation in Xenopus larvae (tadpoles) using fluorescent proteins. Dr. Lemkine headed the museum’s technology-transfer program. “We realized that we could industrialize the amphibian model to efficiently screen large numbers of samples.”

Tadpoles are a recognized model for bioscience research, and their endocrine, cardiovascular, and neurological systems are especially similar to those of humans. WatchFrog designs biological models that predict the impact of chemical agents on human health. They are used for the environmental testing of pollutants and by the pharmaceutical and cosmetic industries.

WatchFrog genetically engineers tadpoles based on individual customer needs. For example, cosmetic companies are interested in highly specific assays rather than assays with high sensitivity. “They want to know with 100 percent certainty if a contaminant is present or not, and they don’t want false positive results,” says Dr. Lemkine. On the other hand, environmental monitoring requires the detection of contaminants at low concentrations, and tadpole systems sense nanogram-per-liter levels of polluting chemicals.

A fluorescent tadpole lights up to reveal endocrine chemicals

Environmental Testing

The fluorescent tadpoles detect how chemical compounds, such as heavy metals or endocrine-disrupting chemicals, impact the embryonic development of vertebrates. The Organization for Economic Co-operation and Development, headquartered in Paris, adopted the tadpole assay as a reference for hormonal disruptors of thyroid function. The same system screens for other toxicity effects in water systems, industrial wastes, cosmetic ingredients, and pharmaceutical manufacturing.

The fluorescent tadpoles provide more than a simple substrate/receptor detection system. Genetically engineered models are “transcriptional assays that look at downstream effects on the genetic targets of the contaminant,” says Dr. Lemkine. The tadpoles can screen for entire signaling pathways induced by a contaminant. The fluorescent response is rapid and visible within hours. Multiple markers and fluorochromes allow the detection of a number of metabolic functions and genes simultaneously.

Along with Paul Johnson, a professor of physics and astronomy at the University of Wyoming, Laramie, WatchFrog invented the Frog Box, used onsite to monitor pollutants at a water treatment facility or in a river downstream from industrial or pharmaceutical plants. The Frog Box, a two liter, double-sided chamber containing tadpoles, is placed in direct contact with the natural water source. When the tadpoles are exposed to pollutants, they fluoresce, and the amount of fluorescence is measured as they swim through a channel that connects the two sides of the Frog Box.

“We sort tadpoles on the basis of their fluorescence similar to how flow cytometers sort cells in the lab,” says Dr. Lemkine. The company has installed prototype Frog Box systems at sites in France and Spain and an experimental station may soon be set up in the U.S.

Other types of commercial toxicity models are based on worms and zebrafish, but these animals are less similar to humans. Tadpoles have metabolic and immune systems closer to humans and a more complex heart and circulatory system than zebrafish. “Amphibians are a natural model for environmental risk assessment,” says Dr. Lemkine, and “in this market we offer a novel solution.”

Pharmaceutical Applications

Pharmaceutical companies, too, can benefit from the tadpole technology to bridge the gap between in vitro screening and preclinical studies. Tadpole-based assays help to identify and optimize molecules and then screen drug candidates before moving on to more costly testing in mice.

Drugs being developed to treat osteoporosis, hormone-dependent cancers, and vascular and neurological disorders are well suited for tadpole screening. Tadpoles also have exceptionally large melanocytes on their skin, making them attractive for investigating the causes of melanoma. Additionally, WatchFrog is creating tadpole models with neuronal lesions that mimic Parkinson disease and multiple sclerosis.

The genetic constructs are tailored to respond to various pharmacological effects. Tadpoles can be supplied in various stages of embryonic development. Large numbers of compounds are screened rapidly by tadpoles placed in 96-well plates. The fluorescent response to a chemical can be tracked in different organs during embryonic development. WatchFrog supplies tadpoles in different embryonic stages to pharmaceutical companies and CROs.

The tadpole assays are easy to perform. A test substance is directly absorbed from solution. The fluorescence is readily visible through the transparent tadpoles. Results are obtained at low cost within a few hours, and a standard fluorimeter measures dose-response relationships. Ethical issues about animal research are avoided because tadpoles are not considered animals.

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