Optimization and prevalidation of the in vitro AR CALUX method to test androgenic and antiandrogenic activity of compounds

Abstract

To date there are no validated methods available to test androgenicity or antiandrogenicity in vitro. A problem with testing androgenicity using reporter genes is the possibility by other steroid receptors than androgen receptors to activate the same reporter gene, thereby lowering selectivity. To avoid this we have established a robust and very selective method, the AR CALUX reporter gene assay, to test androgenic and antiandrogenic activity of compounds in vitro. This assay uses a human U2-OS cell line stably transfected with the human androgen receptor and an androgen receptor responsive reporter gene. We optimized protocols to be used in combination with AR CALUX cells and carried out an in house prevalidation. In addition we successfully transferred this assay to another laboratory, leading to comparable test results with a panel of androgen receptor agonists and antagonists. The assay was able to readily rank a range of chemicals on the basis of their EC50 values. The CALUX assay was found to be selective for androgens and seemed not influenced by signaling through other steroid receptors.

Association of lifestyle and demographic factors with estrogenic and glucocorticogenic activity in Mexican American women

“…CALUX assays are used to identify receptor-mediated signaling pathways of gene expression by specific compounds such as estrogens and androgens (44,45). We relied on these bioassays to agnostically measure ER and GR agonist and antagonist activity profiles.…”

Widespread contamination of coastal sediments in the Transmanche Channel with anti-androgenic compounds

“…The AR CALUX bioassay is a reporter gene assay, comprising a human osteoblast cell line carrying a luciferase gene under transcriptional control of an androgen responsive element. The assay was performed according to published methods (Sonneveld et al, 2005;van der Burg et al, 2010), and androgen receptor activity responses were expressed as relative amounts of reference compounds equivalents; ng dihydrotestosterone (DHT) eq for agonist and lg flutamide (FLU) eq for antagonist activity expressed as per g of dry weight (dw) for sediment or g wet weight (ww) for clam samples. To measure antagonist activity, DHT (0.3 nM) was added to the culture medium.…”

A critique of the EC’s expert (draft) reports on the status of alternatives for cosmetics testing to meet the 2013 deadline

“…expected to go to ECVAM validation. Pre-validation study showed excellent agreement for 14 out of 16 chemicals (Van der Burg et al, 2010). Up to 85% agreement with rabbit test for 50 chemicals (Sonneveld et al, 2011). approaches to waive animal testing, such as exposure-based techniques.…”