Xenoestrogenicity in in vitro assays is not caused by displacement of endogenous estradiol from serum proteins.
The possibility that compounds tested for estrogenicity can compete for binding places on serum proteins and cause an increase of available and very potent endogenous estrogens is of great interest for both the in vitro assay results and the prediction of risk for humans. In in vitro assays, small amounts of estradiol remaining after the charcoal stripping of serum applied in the culture medium could be displaced by the tested compounds, leading to an estrogenic response that might be falsely attributed to the test compound. We have studied the stripping efficiency of charcoal and measured whether reported xenoestrogens can displace estradiol from serum in an in vitro assay using negligible depletion-solid phase microextraction (nd-SPME). Possible competition was also studied with a mathematical exposure model, from which the predictions were compared to the measurements. We found that the common charcoal stripping procedure removed 99% of initially present estradiol. Additionally, our results with charcoal adsorption indicate that charcoal is not useful for serum protein binding assays, as it adsorbs more than the free fraction of ligand. Although the competition model predicted a displacement of estradiol from the serum proteins at the higher applied doses of xenoestrogen, the measurements showed no displacement. Therefore, we conclude that estrogenic responses in the in vitro assay applied here are not caused by displacement of remaining estradiol in the stripped serum. The possibility remains, however, that our displacement hypothesis does apply for estrogen sulfates, as these are present in much higher concentrations than estradiol in stripped serum.
Heringa
Minne B
MB
Institute for Risk Assessment Sciences (IRAS), Utrecht University, Yalelaan 2, 3584 CM Utrecht, the Netherlands.
van der Burg
Bart
B
van Eijkeren
Jan C H
JC
Hermens
Joop L M
JL
D000327:Adsorption; D001665:Binding Sites; D001798:Blood Proteins / Q000378:metabolism*; D002460:Cell Line; D002606:Charcoal / Q000737:chemistry; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism* / Q000493:pharmacokinetics; D004968:Estrogens, Non-Steroidal / Q000145:classification / Q000494:pharmacology* / Q000633:toxicity; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D008954:Models, Biological; D015262:Xenobiotics / Q000145:classification / Q000494:pharmacology* / Q000633:toxicity
D001798:Blood Proteins; D003470:Culture Media; D004968:Estrogens, Non-Steroidal; D007202:Indicators and Reagents; D015262:Xenobiotics; D002606:Charcoal; D004958:Estradiol