Incorporation of a metabolizing system in biodetection assays for endocrine active substances

Abstract

The use of in vitro assays for the biodetection of endocrine active substances (EAS) aims to reduce and replace 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 introducing an S9 metabolic system into the CALUX-reporter gene assays for estrogen receptor α- and anti-androgen receptor-mediated activities was developed. Methoxychlor, which exhibits increased estrogenic and anti-androgenic activities after biotransformation, was used to establish the method in ERα 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 bioactivation potential. 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 S9 resulted in a greatly increased estrogen receptor-mediated transcriptional activation, while the shift in EC50 was only minor. It is likely that both enzyme kinetics and metabolite stability affect the composition of the final metabolite mixture and thus influence the observed effects. Together these results demonstrate the relevance of including biotransformation in in vitro bioassays for the detection of EAS.