E-SCREEN

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E-SCREEN is a cell proliferation assay based on the enhanced proliferation of human breast cancer cells (MCF-7) in the presence of estrogen active substances. The E-SCREEN test is a tool to easily and rapidly assess estrogenic activity of suspected xenoestrogens (singly or in combination). This bioassay measures estrogen-induced increase of the number of human breast cancer cell, which is biologically equivalent to the increase of mitotic activity in tissues of the genital tract. It was originally developed by Soto et al.^[1] and was included in the first version of the OECD Conceptual Framework for Testing and Assessment of Endocrine Disrupters published in 2012. However, due to failed validation, it was not included in the updated version of the framework published in 2018.^[2]

The E-SCREEN test

The E-SCREEN cell proliferation assay is performed with the human

Dulbecco’s modified Eagle’s medium (DMEM) with fetal bovine serum (FBS) and phenol red as buffer tracer (culture medium), at 37 °C, in an atmosphere of 5% CO₂ and 95% air under saturating humidity. To accomplish the E-SCREEN assay the cells are trypsinized and plated in well culture plates. Cells are allowed to attach for 24 h, and the 4 seeding medium is then removed and replaced with the experimental culture medium (phenol red free DMEM with charcoal dextran treated fetal bovine serum -steroid-free-).

The estrogenic activity results are expressed as mean ± standard deviation of the proliferative effect (PE), which represents the maximum proliferation induced by the test compound, and is the ratio between the highest cell number achieved with the sample or 17-β-estradiol and the cell number in the control group:

${\displaystyle \quad PE={(max\ cell\ number)\ sample \over (cell\ number)\ solvent\ control}}$

The estrogenic activity of a sample is evaluated by determining the relative efficacy of stimulation, called the relative proliferative effect (RPE%). The RPE compares the maximum proliferation induced by a sample with that induced by 17-β-estradiol:^[5]

${\displaystyle \quad RPE\%={(PE-1)\ sample \over (PE-1)\ 17\ Beta\ estradiol}\cdot 100}$

The RPE can be used to define full agonists for ER, between 80% and 100% relative proliferation. Partial and weak agonists induce a relative cell proliferation from 25% up to 80%, or 10% to 25%, respectively.

• Shortcomings/Limitations: One of the limitations for determining estrogenicity of chemicals by checking the proliferation of ER positive MCF-7 cell line is that
  mitogens other than estrogens can also influence cell proliferation thus rendering non-specific responses by chemicals.^[5]

1. It is a cell line of endocrine origin and is
   ER positive
   .
2. Expresses
   5α-reductase
   like other steroidogenic cells.
3. Possesses endogenous aromatase activity which converts
   androgens
   to estrogens.
4. Can elicit an estrogen-induced response involving both genomic and non-genomic pathways.

Mechanisms of action

The biological effects of estrogens (and xenoestrogens) are mediated through the estrogen receptor (ER). Estrogen receptors, which belong to a large superfamily of nuclear receptors, are transcription factors that induce transcription of target genes after binding to specific DNA sequences in their promoter.

There are two isoforms (ERα and ERβ):

1. The
   ERα is present in the uterus, and it is thought to drive the uterotropic response, since the uteri of the ERα "knock-out" mice do not respond to the estrogen administration.^[6]
2. The
   ERβ
   is present in organs such as prostate, hypothalamic nuclei and pituitary gland. It is also present, together with ERα in some human breast cancer cell lines.

For example, many xenoestrogens are suspected to contribute to the development of breast cancer in women and prostate and testicular cancers in men, to reduce male fertility and to interact with the immune system.^[7]

Other tests to identify estrogens and/or xenoestrogens

In vivo tests (animal testing)

Uterotrophic Bioassay in Rodents: OECD Test Guideline 440

The gain of the number of human

1. An initial response is an addition of weight owing to water imbibition.
2. This response is succeeded by a weight gain as a result of tissue growth.

The uterus responses in rats and mice can be qualitatively compared.

• Shortcomings/Limitations: The increase of uterine wet weight is not a specific estrogen respond. In addition, this assay has problems inherent to animal testing, is costly and time consuming.^[10]

Vitellogenin assay

Exposure to estrogens (xenoestrogens) may be also easily assessed in fish, reptiles or birds by measuring their vitellogenin (VTG) plasma levels. An example is that expression of the egg yolk precursor protein vitellogenin can be measured by the E-SCREEN test in juvenile brown trout. Usually, vitellogenin is only produced by female fish, because it is estrogen-dependent. Nevertheless, the synthesis of this protein in males and young fish can also occur, as xenoestrogens can also affect the hepatic receptors and activate it. Hence, a frequently used biomarker of exposure to estrogen active substances in the environment is the measurement of vitellogenin levels in male and juvenile trout.^[11]

In vitro test (quantitative bioassays using cells in culture)

Induction of estrogen-related proteins, such as pS2, prolactine or transforming growth factor β-3 (TGF β-3)

In several stable cell lines, it is possible to induce specific estrogen-related proteins. For example, in the human MCF-7 breast cancer cell line the protein pS2 is strongly up-regulated by estrogen. Induction of the pS2 protein can be detected already one hour after estrogen, or a given compound acting as an estrogen, is added to the cells.

Induction of reporter genes under control of estrogen-responsible elements

It is possible to generate an estrogen-responsive reporter cell line by introducing into a situable cell line specific DNA sequences that induce transcription of target genes of a readily measurable protein (the so-called reporter gene; e.g. firefly luciferase). That is an in vitro reporter gene assays detecting estrogen receptor (ER) activation. These bioassays form a group of the so-called CALUX (Chemically Activated LUciferase eXpression) bioassays. These systems are exemplified by the estrogen receptor ER CALUX bioassay consisting of the human T-47D breast tumor cell line expressing estrogen receptors (ER) endogenously together with an ER-specific Luciferase construct. ^[12] A version of the ER CALUX bioassay, consisting of the human U2OS osterosarcoma cell line transfected with both ERα and ER-specific Luciferase construct is currently under validation to be included in OECD Test Guideline 455.^[13]

References