title: Detection of the anti-androgenic effect of endocrine disrupting environmental contaminants using in vivo and in vitro assays in the three-spined stickleback.
abstract: We have previously developed a novel in vitro assay that utilises cultures of primed female stickleback kidney cells for the screening of potential androgenic and anti-androgenic environmental contaminants. Stickleback kidney cells are natural targets for steroid hormones and are able to produce a protein, spiggin, in response to androgenic stimulation. We undertook a combined in vivo/in vitro study where we used the magnitude of spiggin production as an endpoint to test the anti-androgenic properties of the pharmaceutical androgen antagonist flutamide and three environmental contaminants: the organophosphate insecticide fenitrothion, the urea-based herbicide linuron and the fungicide vinclozolin. In vitro, kidney cells were exposed to a range of concentrations [from 10(-14) M (2.5 pg/L) up to 10(-6) M (280 microg/L)] of the test compounds alone for determining agonist activities, or together with 10(-8) M (3 microg/L) dihydrotestosterone (DHT) for determining antagonist activities. An in vivo flow-through aquarium-based study was carried out in parallel. Female sticklebacks were exposed to a range of concentrations of the same chemicals alone or in combination with DHT (5 microg/L) for 21 days. All of the compounds significantly inhibited DHT-induced spiggin production in a concentration-dependent manner in both the in vitro (FN > or = FL > or = LN > VZ) and in vivo (FN > FL > or = VZ > LN) assays. Fenitrothion and flutamide inhibited spiggin production in vitro at a concentration as low as 10(-12) M (P < 0.05), while linuron and vinclozolin inhibited DHT-induced spiggin production at concentrations of 10(-10) M (P < 0.05) and 10(-6) M (P < 0.001) respectively. Similarly, fenitrothion and flutamide were the most potent chemicals in vivo and significantly reduced DHT-induced spiggin production at a concentration of 10 microg/L and 25 microg/L respectively (P < 0.01). Both linuron and vinclozolin induced a significant decrease in DHT-induced spiggin production at a concentration of 100 microg/L when tested in vivo. In addition, kidney cell primary culture was used to test the (anti-)androgenic effects of the major environmental contaminants: oestradiol (E2), nonylphenol (NP) and bisphenol A (BPA) for the first time in teleosts. We observed that these compounds were able to significantly inhibit spiggin production at high doses (E2: 270 microg/L; NP: 2.2 microg/L; BPA: 2.3 microg/L). When tested in the absence of DHT, none of the compounds showed a significant agonistic activity in either in vivo or in vitro assays. Overall, our data further demonstrate that kidney cell primary culture is a reliable and a sensitive screening tool for the detection of (anti-)androgenic compounds. In addition, our study represents the first attempt to develop a combined in vivo/in vitro screening strategy for assessing the effects of (anti-)androgenic endocrine disrupters.
authors.affiliation: University of Bergen, Institute of Biology, HIB, Thormohlensgt. 55, 5020 Bergen, Norway. jolly.cecile@bio.uib.no
authors.forename: Cécile
authors.initials: C
authors.lastname: Jolly
authors.forename: Ioanna
authors.initials: I
authors.lastname: Katsiadaki
authors.forename: Steve
authors.initials: S
authors.lastname: Morris
authors.forename: Nadine
authors.initials: N
authors.lastname: Le Belle
authors.forename: Sylvie
authors.initials: S
authors.lastname: Dufour
authors.forename: Ian
authors.initials: I
authors.lastname: Mayer
authors.forename: Tom G
authors.initials: TG
authors.lastname: Pottinger
authors.forename: Alexander P
authors.initials: AP
authors.lastname: Scott
mesh_terms: D018674:Adrenergic Antagonists / Q000633:toxicity*; D000818:Animals; D002478:Cells, Cultured; D052244:Endocrine Disruptors / Q000633:toxicity*; D005260:Female; D029941:Fish Proteins / Q000032:analysis / Q000235:genetics; D005618:Fresh Water / Q000032:analysis; D005786:Gene Expression Regulation / Q000187:drug effects*; D023701:Smegmamorpha / Q000502:physiology*; D014874:Water Pollutants, Chemical / Q000032:analysis / Q000633:toxicity*
chemical_list: D018674:Adrenergic Antagonists; D052244:Endocrine Disruptors; D029941:Fish Proteins; D014874:Water Pollutants, Chemical; C429543:spiggin