Download summary of the current status of development of non

METI
Ministry of Economy,
Trade and Industry, Japan
SUMMARY OF THE CURRENT STATUS OF
DEVELOPMENT OF NON-ANIMAL TEST METHODS
I.
Receptor Binding Assay / Reporter Gene Assay
1. Outline
1) Receptor Binding Assay
This is an in vitro assay for measuring the extent of the binding affinity between chemicals and
receptors. We have been trying to establish a method for measuring the binding affinity of
the chemicals to estrogen, androgen, or thyroid hormone receptors (ER, AR, and TR)
respectively by measuring the extent of the competition reaction with the reference hormone
labeled with radioisotope (RI). Assay systems of ER alpha of respective species of human,
rat and ER beta of human have been established so far.
2) Reporter Gene Assay
The hormone such as estrogen binds to a hormone receptor in a cell to be formed into a
receptor-hormone complex dimer. In the case of estrogen, the dimer binds to estrogen
response element (receptor dimer binding site) located upstream of the estrogen receptor target
gene and activates the target gene so as to express hormone effects. The reporter gene assay
is an in vitro assay for measuring the transcription activity of the ligand (hormone) dependent
target gene, which is characteristic in nuclear hormone receptors.
For example, in the case of assessing the estrogenic effect of chemicals, plasmid (reporter
plasmid) with reporter genes such as luciferase genes, which would be an indication of
estrogen receptor activation, was constructed downstream of the estrogen response element as
the target gene. When the reporter plasmid is introduced into cells like MCF-7, originally
expressing the estrogen receptors, transcription of the reporter genes is induced, depending on
the extent of the affinity of the chemicals to the receptors. In the case of using luciferase
genes for the reporter gene, the transcriptional activity of the chemicals bound to the estrogen
receptor can be measured by the amount of luciferase protein (namely, expression level of
luciferase activity) as the index.
When using cultured cells expressing no estrogen receptor, the simultaneous introduction of
both plasmid expressing the estrogen receptors and reporter plasmid into the cell enables the
reporter gene assay.
2. Present status
1) Receptor Binding Assay
Human estrogen receptors produced from Escherichia coli by a genetic engineering method
were used for the receptor binding assay with RI-labeled estradiol as a reference ligand.
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2) Reporter Gene Assay
Assay systems using transient cell lines with human ER alpha, human AR or rat ER alpha and
stable cell lines with human ER alpha genes have been established so far. With regard to
human ER beta and human AR, studies to develop a transient or stable cell line respectively
have just started.
Assay system
Receptor
human ER alpha
Transient
expression
system
human ER beta
human AR
rat ER alpha
human ER alpha
Stable clone
human AR
Status
• Showing approximately 10 times the transcriptional
activity relative to 1 nM of E2
(Just started)
• Showing approximately 60 times the transcriptional
activity relative to 10 nM of DHT
• Showing approximately 20 times the transcriptional
activity relative to 1 nM of E2
• Favorable relationship recognized as the result of
comparing 25 chemicals with uterotrophic assays
• Showing approximately 100 times the transcriptional
activity relative to 100 pM of E2
• Confirming stability till 4 months (30 passages)
• Completing data acquisition of 948 compounds
(Just started)
3) Data collection
The data on human ER alpha agonistic activity of approximately 950 chemicals were obtained
by using both the receptor binding assay and the reporter gene assay.
3. Validation of the assay methods
A validation study on a reporter gene assay system using a stable cell line with human ER alpha
has been conducted by CERI (Chemicals Evaluation and Research Institute, Japan) and a
manuscript is now being prepared for publication. The assay system was evaluated for the
relevance and reliability in accordance with the ICCVAM submission guideline (1998). The
relevance of the assay system was evaluated by comparing the EC50 values obtained by our
system with those already published. Reliability of the assay system was evaluated by periodic
analyses of the reproducibility of the responses of the test system to estradiol, testosterone and
bisphenol A over about 4 months. The duration was decided according to the longest subculture
period for one stock tube of the cell line. In addition, the assay performance was evaluated by
“two-by-two table analysis” with reference data obtained by immature rat uterotrophic assays
conducted by CERI.
Similar validation work on the receptor binding assay for human ER alpha is now taking place.
In addition to the above validation study conducted by a single laboratory (CERI), it is desirable
to confirm the validity of the assay system by multiple laboratories. Therefore, METI would
like to discuss how to share the advanced validation works among OECD member countries.
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4. Patent issue
The patent issue inevitably arises in the development of such in vitro assay systems that need the
state-of-the-art knowledge and technology. According to our preliminary investigation into
patents, commercial usage of a reporter gene assay system itself is patented. Besides, several
basic technologies which are necessary to develop a reporter gene assay system, are also patented.
Thus, the patent issue is an important one for discussion.
However, the patent issue appears to be less serious for receptor binding assays because most of
the technology used for the assay method is much simpler than that of reporter gene assays.
II. 3-Dimensional Quantitative Structure-Activity Relationship (3D-QSAR)
1. Outline
To select the chemicals having hormonal effects out of the enormous number of chemicals, we
have been developing a system to predict the binding properties of chemicals to hormone
receptors based on the 3-dimensional structure of a binding complex between the chemical and
the hormone receptor. The binding properties of the chemicals to the hormone receptors are
predicted by the free binding energy to the estrogen receptor calculated from the structure of the
chemicals.
2. Present status
The current objective is to establish a prototype of 3-dimensional structure-activity relationship
(3D-QSAR) model relative to the human ER alpha. The data on the receptor binding assay of
approximately 500 chemicals, which were commercially available, with different substituents
such as hydrogen bond and with different molecular size that may affect the binding of a
chemical to hormone receptors, were used for the validation of 3D-QSAR. However, the
performance in the prediction by the present model is not sufficient for practical use at present.
Thus, we may need to examine a binding prediction method with consideration to the multiple
binding modes (binding state with multiple metastable binding modes), which are thought to be
important for calculating the binding affinity of the weak binder.
III. Future Plan for Development of Non-animal Te st Methods
In addition to continuing the above-mentioned activities, METI currently has a plan for
developing/validating the following non-animal assay systems.
(1) A receptor binding assay for human thyroid hormone receptor (TR)
(2) A reporter gene assay with human TR
(3) A thyroid hormone synthesis assay using a rat cell line (FRTL-5)
(4) An aromatase assay system using ovarian granulosa-like tumor cells (KGN)
(5) Validation of a commercially available reporter gene assay system with an aryl hydrocarbon
receptor (AhR) (CALUXTM)
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