John Sanseverino, Ph.D., Melanie Eldridge, Alice Layton, Jason Yarbrough, T. Wayne Schultz and Gary Sayler, University of Tennessee
The Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) created by the Environmental Protection Agency (EPA) was mandated with developing methods to screen approximately 87,000 chemicals for biological effects on estrogen, androgen, and thyroid hormone systems. As part of this mandate, EDSTAC proposed that EPA develop rapid, high throughput screening systems to assess a compound’s effects on hormonal systems. Recently, two yeast strains, S. cerevisiae BLYES and S. cerevisiae BLYAS, were developed that produce a rapid, measurable bioluminescent signal in response to either an estrogenic or androgenic compound, respectively. The BLYES assay produced a 17β-estradiol standard curve with an EC50 value of 7.3 ± 3.1 x 10-10 M (n = 20) and lower and upper limits of detection of 4.5 x 10-11 to 2.8 x 10-9 M. In the BLYAS assay using dihydrotestosterone as a standard, the lower limit of detection and the EC50 values were 2.5 x 10-9 M and (9.7 ± 4.6) x 10-9 M (n = 17), respectively. In both assays, bioluminescence was observed in as little as 1 hour and data was collected in 3 – 4 hours. A third strain, S. cerevisiae BLYR which produces constant bioluminescence has also been developed to measure toxicity of each chemical, demonstrated by a decrease in bioluminescence. The primary advantages of these bioluminescent assays over colorimetric-based reporter assays include ease of use, efficiency of gathering data, and potential for use of multiple assays per microtiter plate. Further, when combined with appropriate photodetection technology, they may be used for rapid, remote monitoring of industrial and municipal waste effluents carrying hormonal active agents.
Manuscript 6th International Conference on Pharmaceuticals and Enocrine Disrupting Chemicals in Water