title: Incorporation of a metabolizing system in biodetection assays for endocrine active substances.
abstract: The use of in vitro assays is important for the biodetection of endocrine active substances (EAS), reducing and replacing the in vivo studies required for regulatory assessment. However, this approach often fails to take into account the role of biotransformation on the activity of the test substances. A method incorporating an S9 metabolic system into the CALUX-reporter gene assays for estrogen receptor α- and anti-androgen receptor -mediated activities has been developed. Methoxychlor, which is known to exhibit increased estrogenic and anti-androgenic activities after biotransformation, was used to set up the method in ERa and anti-AR CALUX. For the anti-androgenic assay, stanozolol was used as a competing agonist not metabolized by S9. The method was first applied in both agonist and antagonist modes to methoxychlor and bisphenol A, as positive and negative controls, respectively. Then, benzo(a)pyrene and flutamide were also tested for their potential of bioactivation. Co-treatment with S9 successfully increased the ERα agonist and AR antagonist potency of methoxychlor; no change was observed for bisphenol A. Incubation with S9 also enhanced the anti-androgenic activity of flutamide. Interestingly, the metabolism of benzo(a)pyrene by the S9 resulted in an increased estrogen receptor-mediated transcriptional activation; any increase in the potency was only minor. It is likely that both enzyme kinetics and metabolite stability have influenced these effects, which would affect the composition of the final metabolite mixture. Together these results demonstrate the relevance of including biotransformation in in vitro bioassays for the detection of EAS.
authors.affiliation: Chemical Food Safety, Nestlé Research Centre, Lausanne, Switzerland.
authors.forename: Julie
authors.initials: J
authors.lastname: Mollergues
authors.affiliation: BioDetection Systems, Amsterdam, Netherlands.
authors.forename: Barbara
authors.initials: B
authors.lastname: van Vugt-Lussenburg
authors.affiliation: OFI - Austrian Research Institute for Chemistry and Technology, Vienna, Austria.
authors.forename: Christian
authors.initials: C
authors.lastname: Kirchnawy
authors.affiliation: OFI - Austrian Research Institute for Chemistry and Technology, Vienna, Austria.
authors.forename: Reka Anna
authors.initials: RA
authors.lastname: Bandi
authors.affiliation: BioDetection Systems, Amsterdam, Netherlands.
authors.forename: Rosan B
authors.initials: RB
authors.lastname: van der Lee
authors.affiliation: Chemical Food Safety, Nestlé Research Centre, Lausanne, Switzerland.
authors.forename: Maricel
authors.initials: M
authors.lastname: Marin-Kuan
authors.affiliation: Chemical Food Safety, Nestlé Research Centre, Lausanne, Switzerland.
authors.forename: Benoit
authors.initials: B
authors.lastname: Schilter
authors.affiliation: Chemical Food Safety, Nestlé Research Centre, Lausanne, Switzerland.
authors.forename: Karma C
authors.initials: KC
authors.lastname: Fussell
keywords:  biodetection; CALUX® assay ; S9 fraction ; endocrine active substances; metabolism