Antiestrogen assay

The cells are grown in 96-well plates in estrogen-free medium (phenol red free, with charcoal-stripped calf serum) and contain test compounds and antiestrogens at concentrations that are varied over several log orders. For the antiestrogen assay, to the cells, a range of concentrations of samples are added concurrently with I-nM antiestrogens. After grown for 3 days, to determine AP activity, the cells are frozen, defrosted, and incubated with p-nitrophenylphosphate at room temperature, and the hydrolysis product p-nitrophenol is measured kinetically at 405 nm [50]. 

In the 3-day uterotrophic bioassay in mice, a significant increase in wet uterine weights of mice was observed in animals treated with 60 or 120 mg per animal (average animal weight not stated) of an alcohol extract of wild carrot. In comparison to estradiol-17P, the activity was characterized as "extremely weak" (Sharma et al. 1976). In the 3-day antiestrogenic assay, the same extract and doses significantly inhibited the uterotrophic effect of estradiol-17p (Sharma et al. 1976). 

Type II antiestrogens, pure antiestrogens, or selective estrogen receptor down-regulators (SERDs) (Howell et al. 2004b) have no estrogen-like properties in laboratory assays. 

In the E-Screen bioassay, LAS was not effective in promoting cell proliferation (Table 7.3.3). This compound was tested at concentrations of up to 100 pM with no evidence of cellular toxicity. The antiestrogenic effect of this compound was also measured but all samples tested were negative. Because it has been suggested that surfactants of the alkylbenzene sulfonate type are readily degradable and transformed into sulfophenyl carboxylates or SPCs, an important number of SPCs were assayed in the E-Screen test. These SPCs did not induce cell proliferation of MCF7 cells. 

McKim JM, Wilga PC, Breslin WJ, Plotzke KP, Gallavan RH, Meeks RG (2001) Potential estrogenic and antiestrogenic activity of the cyclic siloxane octamethylcyclotetrasiloxane (D4) and the linear siloxane hexamethyldisiloxane (HMDS) in immature rats using the uterotrophic assay. Toxicol Sci 63 37-46... 

Okubo T, Suzuki T, Yokoyama Y, Kano K, Kano I (2003) Estimation of estrogenic and antiestrogenic activities of some phthalate diesters and monoesters by MCF-7 cell proliferation assay in vitro. Biol Pharm Bull 26 1219-1224... 

Assays are expected to detect antiestrogens, but this was not established during the validation process since no estrogen receptor antagonists were tested. 

The estrogenic and antiestrogenic activities of several PBDE congeners and three hydroxylated PBDEs were tested in vitro using human breast cell line assays based on estrogen receptor (ER)-dependent luciferase reporter gene expression (Mccrts et al. 2001). The hydroxylated PBDEs,... 

Fig. 16. Basal ERp transcriptional activation by SRC-1 is AF-2 independent. (A) Dose-dependent activation of ERa and ERP by SRC-1 in absence of Eg. Cos-1 cells were transfected with ERETKLuc reporter along with ERa or ERP and increasing amounts of SRC-1 expression plasmids. Luciferase activities were normalized with P-Gal expression and results are expressed as factor by which response exceeds basal levels (-) and represent the mean SEM of three independent experiments. (B) Pure antiestrogen EM-652 but not the mixed antagonist 4-hydroxytamoxifen (OHT) inhibits basal ERP transcriptional activation by SRC-1. Cos-1 cells were transfected with ERETKLuc along with equivalent amounts of ERP and SRC-1 expression plasmids and incubated with increasing amounts of OHT or EM-652 prior to being assayed for luciferase activity. The maximal induction by SRC-1 alone (solid bar) was defined as 100%. Basal level in absence of SRC-1 is indicated by an open bar. (C) Basal activity of an ERP AF-2 mutant is induced by SRC-1. Cos-1 cells were transfected with ERETKLuc reporter and equivalent amounts of ERP or ERP L509A AF-2 mutant and SRC-1 (solid bars) expression plasmids. Cells were then treated with 10 nM Eg (striated bars) or left untreated (open and solid bars) for 16 hours prior to harvest. Results are plotted as factor by which induction exceeds basal levels (Tremblay et al, 1999).

Most hormone-sensitive cancers will express hormone receptors that can be assayed on biopsy specimens. This allows the clinician to predict whether an individual patient is likely to benefit from hormonal therapy. For example, it is now standard to measure estrogen receptor (ER) and progesterone receptor (PR) content in breast cancer tissue. Patients with ER- or PR-positive tumors are more likely to respond to antiestrogen therapy compared with patients who lack these hormone receptors. 

Although oral administration of 3,000 mg/kg/day unleaded gasoline to female mice for 3 days resulted in a three-fold increase in estrogen metabolism in isolated hepatocytes, there were no functional antiestrogenic effects as assessed by uterotrophic assays (Standeven et al. 1994b). 

In a typical assay, tamoxifen is used as a positive control, and this yields an antiestrogenic response with an IC50 of about 0.324 /rg/ml (575nM). In addition, as with other cell lines, cytotoxicity can be assessed to establish specificity. In general, follow-up work is performed in which competitive binding is assessed with the anti-estrogen (tamoxifen) binding site (43). 

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