Validation of in vitro screening models for progestagenic activities: Inter-assay comparison and correlation with in vivo activity in rabbits Edwin Sonneveld a,1 , Bart Pieterse a , Willem G. Schoonen b , Bart van der Burg a,⇑ a BioDetection Systems BV, Science Park 406, 1098XH Amsterdam, The Netherlands b Department of Toxicology and Drug Disposition, MSD, Oss, The Netherlands article info Article history: Received 17 June 2010 Accepted 26 November 2010 Available online 2 December 2010 Keywords: Validation Progesterone Progesterone receptor Reporter gene assay Screening model McPhail assay abstract The PR CALUX Ò cell line is a stably transfected human U2-OS cell line expressing the human PR and a luciferase reporter construct containing three progesterone-responsive elements coupled to a minimal promoter. The validity of this assay has been studied as an alternative to the McPhail assay in rabbits, an in vivo assay to detect progestins. The PR CALUX assay was characterized by its stable expression of PR protein which leads to induction of endogenous PR target genes by progestins. It was found to have a highly selective response to low levels of different progestins, as well as an insignificant response to other nuclear hormone receptor ligands. As an important step in their validation, the PR CALUX bioassay was compared with another earlier described in vitro bioassay, a Chinese Hamster Ovary (CHO) cell-based PR-CHO reporter gene assay as well as with an in vitro PR-binding (PR-BIN) assay, and the in vivo McPhail assay. This was done using 35 (with the most accurate potency determinations in all tests) and 50 (with less reliable potency determinations in some tests) compounds tested in all assays. The correlation scores between PR CALUX and PR-CHO were r 2 = 0.77, and 0.93, respectively; between PR CALUX and PR-BIN r 2 = 0.69 and 0.80. Comparison between either the PR CALUX or the PR-CHO transactivation assay and the in vivo McPhail assay revealed very good correlations of r 2 = 0.68 (n = 35), and 0.85 (n = 50). The trans- activation assays can discriminate very potent, from potent, weak and inactive compounds rather easily. Besides testing the biological activity of pure chemicals and pharmaceuticals in vitro, the PR CALUX and PR-CHO transactivation assays proved to be relatively good predictors of in vivo progestagenic activity, allowing the use of these assays as prescreening methods or in vitro alternatives. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Progesterone is a lipophilic cholesterol-derived steroid hor- mone and an essential regulator of the growth and development of the female reproductive systems (Graham and Clarke, 1997). Progestins can induce endometrium development in rabbits after a pre-treatment with estrogen (McPhail, 1934). Progesterone is mainly produced in the ovary, but is also secreted by the adrenal cortex, or can be converted from peripheral precursors. Progestins are synthetic compounds which are widely used in female contra- ception, hormone replacement therapy, treatment of hormone- dependent cancer and treatment of reproductive disorders like endometriosis and uterine adenomyosis (Spitz and Chwalisz, 2000). Progesterone activates germinal vesicle breakdown and meiosis, the initiation phase for ovulation. Moreover, progesterone supports pregnancy, while the major clinically exploited effects of progestins in humans are to transform the endometrium into the decidua and to inhibit further ovulation via the inhibition of the GnRH burst from the hypothalamus, leading to the LH surge from the pituitary. Although progestins possess progesterone-like activ- ity, they differ widely from progesterone in their chemical struc- tures, potencies and pharmacokinetics (Stanczyk, 2003). The effects of progesterone in target cells are mediated by the progesterone receptor (PR), a member of the nuclear hormone receptor superfamily, which includes receptors for steroids, reti- noids, vitamin D3 and thyroid hormones (Mangelsdorf et al., 1995). PR is a ligand-dependent transcription factor that regu- lates specific gene expression by binding to specific hormone responsive elements (HREs) within the regulatory DNA sequences of many progesterone-responsive genes. The human PR has at least two distinct isoforms, PR-A and PR-B, of which PR-A lacks the N-terminal 164 amino acids. In general, of both isoforms PR-B has the strongest transcriptional activity, while PR-A can function as a transcriptional repressor. PR-A is activated by sim- ilar ligands, but at a lower level (Schoonen et al., 1998b). Proges- tins bind and transactivate the PRs resulting in activation or repression of gene expression in target cells in a similar manner 0887-2333/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2010.11.018 ⇑ Corresponding author. Tel.: +31 204350750; fax: +31 204350757. E-mail address: Bart.van.der.Burg@bds.nl (B. van der Burg). 1 Present address: Dutch Childhood Oncology Group (SKION), Den Haag, The Netherlands. Toxicology in Vitro 25 (2011) 545–554 Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit