Rapid Fluorescent Detection of (Anti)androgens with spiggin-gfp Medaka

• Published in: Environmental science & technology 2014-09, Vol.48 (18), p.10919-10928
• Main Authors: Sébillot, Anthony, Damdimopoulou, Pauliina, Ogino, Yukiko, Spirhanzlova, Petra, Miyagawa, Shinichi, Du Pasquier, David, Mouatassim, Nora, Iguchi, Taisen, Lemkine, Gregory F, Demeneix, Barbara A, Tindall, Andrew J
• Format: Article
• Language: English
• Subjects: Androgen Antagonists - metabolism; Androgens; Animals; Animals, Genetically Modified; Base Sequence; Cloning, Molecular; Environmental impact; Environmental monitoring; Fish Proteins - metabolism; Fluorescence; Gasterosteus aculeatus; Green Fluorescent Proteins - metabolism; Health hazards; Humans; Molecular Sequence Data; Oryzias - genetics; Oryzias - metabolism; Oryzias latipes; Pesticides; Promoter Regions, Genetic - genetics; Receptors, Androgen - metabolism; Smegmamorpha
• ISSN: 0013-936X; 1520-5851
• DOI: 10.1021/es5030977

Widespread environmental antiandrogen contamination has been associated with negative impacts on biodiversity and human health. In particular, many pesticides are antiandrogenic, creating a need for robust and sensitive environmental monitoring. Our aim was to develop a sensitive and specific transgenic medaka (Oryzias latipes) model bearing an androgen responsive fluorescent reporter construct for whole organism-based environmental screening of pro- and antiandrogens. We analyzed the 5′ regions of the androgen responsive three-spined stickleback (Gasterosteus aculeatus) spiggin genes in silico, revealing conserved blocks of sequence harboring androgen response elements. Identified putative promoters were cloned upstream of GFP. Germinal transgenesis with spg1-gfp led to stable medaka lines. GFP induction was exclusive to the kidney, the site of spiggin protein production in sticklebacks. Significant GFP expression was induced by three or four-day androgen treatment of newly hatched fry, but not by estrogens, mineralocorticoids, glucocorticoids or progestogens. The model responded dose-dependently to androgens, with highest sensitivity to 17MT (1.5 μg/L). In addition to flutamide, the biocides fenitrothion, vinclozolin and linuron significantly inhibited 17MT-induced GFP induction, validating the model for detection of antiandrogens. The spg1-gfp medaka model provides a sensitive, specific, and physiologically pertinent biosensor system for analyzing environmental androgen activity.