Oestrogen reporter transgenic medaka for non-invasive evaluation of aromatase activity.
Vertebrate reproduction involves complex steroid hormone interplay and inter-conversion. A critical element in maintaining sex steroid levels is the enzyme aromatase (cytochrome P450 19A1) which converts androgens to oestrogens. In turn oestrogen signalling is targeted by numerous chemicals, from pharmaceuticals to agricultural chemicals, both frequent sources of contamination in waste waters and consequently rivers. Although many models are now available to address disruption of oestrogen signalling, there are currently no published protocols allowing discrimination between alterations in testosterone metabolism and in oestrogenic signalling. It was with this limitation in mind that we optimised this protocol. We show using a 48h protocol that pre-feeding fry of the choriogenin h-gfp (chgh-gfp) medaka line are sensitive to 0.05nM EE2 (15ng/L), within the range of the lowest published observable physiological effect concentrations for medaka. In addition, co-treatment with testosterone can reveal potential effects of test substances on aromatase enzymatic activity. As the measurements are visualised in real-time without affecting embryo viability, repeated measures are possible. We demonstrate the ability of this model to detect oestrogen receptor agonists, aromatisable androgens, P450 aromatase activity modulators and selective oestrogen response modulators. Importantly, the range of this assay is physiologically relevant.
WatchFrog S.A., 1 rue Pierre Fontaine, 91000 Evry, France; CNRS UMR 7221, Evolution des Régulations Endocriniennes, Département Régulations, Développement et Diversité Moléculaire, Muséum National d'Histoire Naturelle, Paris, France.
Petra
P
Spirhanzlova
WatchFrog S.A., 1 rue Pierre Fontaine, 91000 Evry, France.
Mathilde
M
Leleu
WatchFrog S.A., 1 rue Pierre Fontaine, 91000 Evry, France.
Anthony
A
Sébillot
WatchFrog S.A., 1 rue Pierre Fontaine, 91000 Evry, France. Electronic address: lemkine@watchfrog.fr.
Gregory F
GF
Lemkine
Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan.
Taisen
T
Iguchi
CNRS UMR 7221, Evolution des Régulations Endocriniennes, Département Régulations, Développement et Diversité Moléculaire, Muséum National d'Histoire Naturelle, Paris, France.
Barbara A
BA
Demeneix
WatchFrog S.A., 1 rue Pierre Fontaine, 91000 Evry, France.
Andrew J
AJ
Tindall
D000818:Animals; D030801:Animals, Genetically Modified; D001141:Aromatase / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004967:Estrogens / Q000494:pharmacology*; D009990:Oryzias / Q000235:genetics; D014874:Water Pollutants, Chemical / Q000494:pharmacology
D004967:Estrogens; D014874:Water Pollutants, Chemical; D001141:Aromatase
Androgen; Aromatase; Choriogenin; Endocrine disruption; GFP; Oestrogen; Oryzias latipes; Steroidogenesis