In Vitro Assays for Endocrine Disruptor
Estrogen receptor binding assay using rat uterus cytosol (OPPTS 890.1250)
The estrogen receptor binding assay measures the ability of a radiolabeled ligand ([3H]-17β-estradiol) to interact with the ER in the presence of increasing concentrations of a test item. Rat uterine cytosol is the source of ERα. Rat uterine cytosol is incubated with increasing concentrations of test item and an aliquot of [3H]-17β-estradiol ([3H]-E2). If the test item interacts with the receptor’s hormone binding domain, less [3H]-E2 will be bound resulting in a decrease of receptor-bound [3H] E2 at increasing test item concentrations. If the test item does not interact with the receptor, [3H]-E2 cannot be displaced resulting in a stable amount of receptor bound [3H]-E2. The test item is therefore considered to have negligible estrogen binding activity.
Freyberger-Wilson in vitro estrogen receptor (ERα) binding assay using full length human recombinant ERα (OECD 493)
The Freyberger-Wilson in vitro ER binding assay measures the ability of the radiolabeled ligand [3H]17β-estradiol ([3H]-E2) to bind with the ER in the presence of increasing concentrations of a test item (i.e., competitor). Test items that possess a high affinity for the ER compete with the radiolabeled ligand at a lower concentration as compared with those chemicals with lower affinity for the ER. The test method consists of two major components: a saturation binding experiment to characterize receptor-ligand interaction parameters, followed by a competitive binding experiment (three non-concurrent runs) to characterize the competition between a test item and [3H]-E2 for binding to the ER. The extent of displacement of the radiolabeled estradiol is used to characterize the test item as a binder, non-binder, or generating an equivocal response.
Estrogen receptor transcriptional activation assay (human cell line, HeLa-9903) (OPPTS 890.1300, OECD 455)
The hERα-HeLa-9903 cell line is an estrogen responsive stable cell line derived from a human cervical tumor stably transfected with a human ERα expression vector and a firefly luciferase reporter vector bearing five tandem repeats of a vitellogenin Estrogen-Responsive Element (ERE) driven by a mouse metallothionein (MT) promoter TATA element. This cell line is able to measure the ability of a test item to induce hERα-mediated transactivation of luciferase gene expression.
Androgen receptor binding using rat prostate cytosol (OPPTS 890.1150)
The androgen receptor binding assay measures the ability of a radiolabeled ligand ([3H]-R1881) to interact with the androgen receptor (AR) in the presence of increasing concentrations of a test item. Rat prostate cytosol is the source of AR. Rat prostate cytosol is incubated with increasing concentrations of test item and an aliquot of [3H]-R1881. If the test item interacts with the receptor, less [3H]-R1881 will be bound resulting in a decrease of receptor-bound [3H]-R1881 at increasing test item concentrations. If the test item does not interact with the receptor, it cannot displace [3H]-R1881 resulting in a stable amount of receptor bound [3H]-R1881.
Stably transfected human androgen receptor transcriptional activation assay (OECD 458)
The AR-EcoScreen™ cell line is an androgen responsive stable cell line derived from a CHO-K1 cell line stably transfected with human AR expression vector and a firefly luciferase reporter vector bearing four tandem repeats of androgen responsive element (ARE) from prostate C3 gene-responsive element driven by a minimal heat shock protein promoter. In addition, a Renilla luciferase reporter construct under the SV40 promotor, stably and non-inducibly expressed is transfected as to distinguish pure antagonism from a cytotoxicity-related decrease of luciferase activity.
Steroidogenesis assay (human cell line H295R) (OPPTS 890.1550, OECD 456)
The steroidogenesis assay uses the human adrenocortical carcinoma cell line H295R, which has the ability to produce steroid hormones found in the adult adrenal cortex. Human H295R cells are cultured and subsequently exposed for 48 hours to the test item at seven different non-cytotoxic concentrations, or the reference items prochloraz or forskolin. Subsequently, the concentration of the hormones testosterone and estradiol in the medium will be determined using commercially available ELISA kits or LC-MS. Vehicle controls are included in each assay. Testosterone and estradiol concentrations will be expressed as changes relative to the vehicle control.
Aromatase assay (human recombinant) (OPPTS 890.1200)
The aromatase assay measures the conversion of androgen to estrogen in microsomes containing the aromatase (CYP19) and cytochrome P450 reductase complex. Radioactive substrate (Androst-4-ene-3,17-dione, [1β-3H (N)]) and NADPH will be incubated with microsomes containing the aromatase (CYP19) and reductase complex in the presence or absence of test item or positive control inhibitor (4-hydroxy-androstenedione, OH-ASDN). During the conversion of the radioactive substrate [1β-3H (N)] to estrone, 3H2O is released which is quantified using liquid scintillation counting (LSC) as a direct measurement of aromatase activity per unit reaction time. Competitive inhibition of aromatase activity by a test item can be detected by serial reaction tubes containing increasing concentrations of the test item.
The possible inhibitory properties of a test item for human aromatase activity will be determined in three independent assays. Within each assay, the test item will be tested at eight concentrations in triplicate.