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  • In the present study we determined concentration

    2020-11-30

    In the present study, we determined concentration–response relationships for selected synthetic progestogens using in vitro luciferase transactivation assays driven by PR, ARα and ARβ from Murray–Darling rainbowfish () and compared the profiles with those obtained from similar assays based on human PR and AR. To facilitate the analysis of a wide range of compounds and concentrations, we developed stably transfected cell lines expressing rainbowfish nuclear receptors and hormone-responsive luciferase reporter genes (details can be found in Supplementary methods). This was achieved using dual Mycophenolic acid selection for a geneticin-resistant expression vector (Invitrogen pcDNA3.1) and a hygromycin-resistant luciferase reporter vector (Promega pGL4.26) containing multiple copies of an appropriate response element for each receptor. After selection and clonal expansion, clones were screened for the desired characteristics – rapid growth, high luciferase induction in response to reference compounds relative to untreated controls, and median effective concentrations (EC50) for reference compounds within acceptable range for each receptor. Using this approach, we developed reliable and sensitive hormone-responsive reporter cell lines for rainbowfish PR (rfPR), rainbowfish ARα (rfARα) and rainbowfish ARβ (rfARβ). The resulting reporter cell lines can be considered comparable with commercially available CALUX in vitro bioassays (BioDetection Systems, Netherlands) because they are based on the same cell line (U-2 OS) and utilize similar expression and reporter vectors (, , , , ). To compare the responses of rainbowfish receptors with the corresponding human receptors, CALUX bioassays based on human PR and AR were obtained from BioDetection Systems and validated in our laboratory (). To allow direct comparison of concentration–response profiles, we treated all five reporter cell lines with the same set of progestins and reference compounds: the 19-nortestosterone-derived progestins ETG, GSD, NGM, LNG and 19-NE, the spironolactone derivative, DPN, the mammalian endogenous progestogen, P4, the fish endogenous progestogen, DHP, and the androgen 5α-dihydrotestosterone (DHT). DHT is becoming recognized as an important androgen in some fish species (, ), and we have shown in a previous study using transient transactivation assays that DHT is a functional agonist of rainbowfish ARα and ARβ, displaying potency comparable to the natural ligand, 11-ketotestosterone (11-KT) (). We therefore used DHT as the androgenic reference compound in all three AR-driven reporter assays employed in the present study. To establish concentration–response profiles, cells were seeded into 96-well plates and exposed to test compounds essentially as previously described (). Briefly, after incubating overnight in a growth medium prepared using dextran-coated charcoal-stripped serum to reduce background steroid hormone levels, cells were exposed to various concentrations of test compounds. Dilutions of test compounds were prepared in DMSO prior to adding to the growth medium. Cells were exposed in triplicate wells for each concentration of test compound including solvent controls, and the final solvent concentration was 0.1% in all wells. Luciferase assays were conducted after 24h exposure to test compounds under normal growth conditions (humidified atmosphere at 37°C, 5% CO). Concentration–response profiles are presented in . EC50 values and potencies relative to reference compounds are shown in . As expected, all synthetic progestogens tested were potent agonists of hPR (A). All 19-nortestosterone-derived progestins were more potent hPR agonists than the endogenous ligand, P4, while the spironolactone-derived progestin, DPN, was slightly weaker than P4. In contrast, none of the progestins tested were strong agonists of rainbowfish nuclear PR (rfPR), with only DPN and GSD displaying weak activity (approximate EC50>10M) and all others exhibiting induction factors less than 1.5-fold relative to control and lacking discernible concentration–response relationships at the concentrations tested (up to 10M; B). According to a phylogenetic analysis, rfPR is representative of other teleost PRs, falling within a clade that also includes medaka and perch PRs (Supplementary Fig. 1), and can hence, like fmPR () be considered a typical teleost PR. P4 was only a weak agonist of the rainbowfish PR, which is consistent with the reported ligand selectivity of zebrafish PR as defined using in vitro reporter gene assays ().