Determining relative estrogenicity by quantifying vitellogenin induction in rainbow trout liver slices.
Precision cut tissue slices, by modeling the entire organ, are a valuable tool for studying protein induction or inhibition by test chemicals. This manuscript describes parameters to quantify relative estrogenicity of chemicals in rainbow trout liver slices by measuring vitellogenin (Vg) induction, a well-characterized biomarker of estrogen receptor signal transduction. Hank's medium (phenol-red free) supplemented with Hepes, sodium bicarbonate, and 1% bovine serum albumin was utilized. The experimental parameters were optimized using 1000 nM 17beta-estradiol, a potent estrogen in rainbow trout that induces Vg production in vivo. The addition of trout serum and retention of the media was essential, probably to allow for the accumulation of Vg in the slices and media. Histological examination and ATP analyses indicated no toxicity in control or 17beta-estradiol-treated liver slices after 120 h. Induction was 4-fold greater with 25% serum containing media compared to media with 10% serum. We observed Vg induction as great as 500-fold over controls at 96 h in liver slices and media containing 25% serum and 1000 nM 17beta-estradiol. Controls without 17beta-estradiol, incubated in media with 10 or 25% serum, exhibited no detectable Vg production, indicating that the induction seen was not from the media or serum. We observed that 48 h was required for significant Vg induction in the media and liver slices. Maximum induction in slices occurred at 96 h, whereas media Vg levels continued to increase to 120 h, suggesting a time delay between Vg production and excretion by the liver. The feasibility of this model to detect weak environmental estrogens was determined with 0-250 microM o,p'DDE and bisphenol A. Both compounds induced Vg in this model with EC50 values of 10(4) and 2x10(5) higher than E(2), respectively. Our results indicate the importance of media, serum, and time selection for optimal Vg induction. This model allows for the determination of relative estrogenicity of chemicals in a controlled in vitro system while utilizing the advantages of precision cut slice technology.
Shilling
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Department of Environmental and Molecular Toxicology and Marine/Freshwater Biomedical Sciences Center, Oregon State University, Corvallis, Oregon 97331, USA.
Williams
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001559:Benzhydryl Compounds; D003634:DDT / Q000494:pharmacology; D003633:Dichlorodiphenyl Dichloroethylene / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology*; D004965:Estrogen Antagonists / Q000494:pharmacology; D004968:Estrogens, Non-Steroidal / Q000494:pharmacology*; D005260:Female; D066298:In Vitro Techniques; D007306:Insecticides / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D010636:Phenols / Q000494:pharmacology; D013629:Tamoxifen / Q000494:pharmacology; D014819:Vitellogenins / Q000096:biosynthesis*; D000098826:Bisphenol A Compounds
D000255:Adenosine Triphosphate; D001559:Benzhydryl Compounds; D003634:DDT; D003633:Dichlorodiphenyl Dichloroethylene; D004958:Estradiol; D004965:Estrogen Antagonists; D004968:Estrogens, Non-Steroidal; D007306:Insecticides; D010636:Phenols; D013629:Tamoxifen; D014819:Vitellogenins; C006780:bisphenol A; D000098826:Bisphenol A Compounds