Environmental News Zebrafish assay detects endocrine disrupters A new bioassay for estrogens using transgenic zebrafish could help screen chemicals for estrogenic effects. The tool, developed by researchers at the Netherlands Institute for Developmental Biology in Utrecht, should also improve scientists' understanding of the mechanisms of action behind endocrine disruption {Environ. Sci. Technol. 2000, 34 (20)) 44394444). Tests currently being validated for the screening of endocrinedisrupting chemicals in the
United States and the European Union can be divided into in vitro systems working on the cellular or molecular level and traditional in vivo systems using and sacrificing whole animals. The new method ideally complements existing in vitro screening methods, claims project leader Bart van der Burg, because it can be used to rule out false positives. In contrast to in vitro systems, the zebrafish assay takes into account bioavailability and metabolism of estrogens and can be used for various developmental fish stages. This Reporter gene induction in transgenic zebrafish method is also easier to conduct Transgenic zebrafish contain a luciferase reporter gene (Luc) and less time-concoupled to an estrogen binding sequence. Estrogens bind to the suming than tradiestrogen receptors; the resulting complex dimerizes; and these tional in vivo tests. bind to estrogen response elements (ERE), including those present To transform on the introduced pERE-TATA-Luc construct. Transcription and zebrafish into potranslation of luciferase are induced, which can then be quantified tential biosensors by a light-emitting reaction. for endocrine disruption, doctoral student Juliette Legler introduced an estrogen-binding sequence linked to a TATA box (see figure) as a promoter together with a luciferase reporter gene into the fish DNA. When estrogenic substances bind to the DNA, the luciferase gene is transcribed. The luciferase protein can easily be quantified by measuring light activity after the enzyme substrate, luciferin, is introduced. Although thorough validation is Source: Ferdinand Vervoor Deldonk, Netherlands Institute for Developmental Biology. still necessary to 4 5 0 A • NOVEMBER 1, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS
judge the method's full potential, it represents a significant step forward in environmental monitoring, says fish ecotoxicologist John Sumpter of Brunei University in Uxbridge, U.K. He envisions that one day researchers might be able to use transparent fish and green fluorescent protein as a reporter, enabling live detection of organ-specific reactions in the fish and thereby helping to reduce the number of animals used for in vivo studies. Rik Eggen, molecular ecotoxicologist at the Swiss Federal Institute for Environmental Science and Technology, in Diibendorf, Switzerland, sees an added benefit in the transgenic zebrafish's potential to help explain mechanistically how the complex cascade of events leading to endocrine disruption works. Because effects can be assessed on the tissue level, the tool has already led to the discovery that the testis is the most responsive target tissue to estrogens in male adult zebrafish. Future research planned by van der Burg's group will concentrate on a full validation of the zebrafish bioassay and the development of different fish lines to check for other groups of hormones important in endocrine disruption, such as androgens, to complement the investigations on estrogenic effects. Researchers studying endocrine disruption, however, should not concentrate too much on screening large numbers of chemicals in laboratory assays, Sumpter says. "We are at the same stage here as we were in global warming 10 years ago," he states. "What we now need to know is if endocrine disruption is a significant factor impacting humans and wildlife on the population level." —ANKE SCHAEFER © 2000 American Chemical Society
