Development of a stickleback kidney cell culture assay for the screening of androgenic and anti-androgenic endocrine disrupters.
Issues raised by the presence in the environment of chemicals able to mimic or antagonize the action of androgenic hormones are of growing concern. Here we report the development of a novel in vitro test for the screening of (anti-)androgenic chemicals, based on primary cultures of stickleback kidney cells that produce a protein, the spiggin, in response to androgenic stimulation. Cell spiggin content was measured by ELISA. Comparison between cell cultures from quiescent males, photoperiodically stimulated males, control females and dihydrotestosterone (DHT)-primed females led to the selection of cell cultures from DHT-primed females for the development of a standardized protocol. 48h of treatment with androgens proved to be sufficient to induce concentration-dependent increase in spiggin cell content with a high sensitivity. DHT induced a significant spiggin increase at 10(-12)M, while testosterone (T) and the teleost specific androgen 11-ketotestosterone (11-KT) had a significant effect at 10(-10)M. Maximal responses were obtained with 10(-8)M DHT and 10(-6)M T and 11-KT. This indicates a higher sensitivity to DHT than to T and 11-KT, in agreement with previous data on stickleback kidney androgen receptor affinity. No effect was observed with other steroids or thyroid hormone, indicating the androgen specificity of the test. The anabolic steroid 17beta-Trenbolone (TB) was able to stimulate spiggin synthesis in a concentration-dependent manner with a significant effect at a concentration as low as 10(-10)M, and a maximal effect at 10(-6)M. The synthetic human androgen receptor antagonist, flutamide had no effect alone, but concentration-dependently inhibited the stimulatory effect of 10(-8)M 11-KT with a complete inhibition at 10(-6)M flutamide. This cell culture system provides an innovative tool for the rapid and sensitive screening of androgenic and anti-androgenic properties of environmental contaminants.
Jolly
Cécile
C
Department of Biology, University of Bergen, Institute of Biology, HIB, Thormohlensgt. 55, 5020 Bergen, Norway. jolly.cecile@bio.uib.no
Katsiadaki
Ioanna
I
Le Belle
Nadine
N
Mayer
Ian
I
Dufour
Sylvie
S
D045930:Anabolic Agents / Q000633:toxicity*; D000726:Androgen Antagonists / Q000633:toxicity*; D000728:Androgens / Q000633:toxicity*; D000818:Animals; D002478:Cells, Cultured; D013196:Dihydrotestosterone / Q000633:toxicity; D005260:Female; D029941:Fish Proteins / Q000032:analysis / Q000096:biosynthesis / Q000187:drug effects; D005485:Flutamide / Q000633:toxicity; D015870:Gene Expression / Q000187:drug effects; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008297:Male; D023701:Smegmamorpha / Q000378:metabolism*; D013739:Testosterone / Q000031:analogs & derivatives / Q000633:toxicity; D013997:Time Factors; D018675:Toxicity Tests / Q000379:methods*; D014204:Trenbolone Acetate / Q000633:toxicity
D045930:Anabolic Agents; D000726:Androgen Antagonists; D000728:Androgens; D029941:Fish Proteins; C429543:spiggin; D013196:Dihydrotestosterone; D013739:Testosterone; D005485:Flutamide; C003600:11-ketotestosterone; D014204:Trenbolone Acetate