JP2003259895A - Method for detecting endocrine disrupter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an endocrine disruptor-measuring technique which uses as an indicator the change in the aromatase activity of a biosystem reflecting a state near to a living environment. <P>SOLUTION: This method for detecting a test substance as an endocrine disruptor with an aromatase activity as an indicator is characterized by comprising a process for culturing a cell strain having the aromatase activity in the presence or absence of the test substance under physiological conditions and measuring the aromatase activity of the cultured cells, and a process for detecting the change in the aromatase activity of the cell strain cultured in the presence of the test substance, while using the aromatase activity of the cell strain cultured in the absence of the test substance as a standard, thereby specifying the test sample giving the change. And a kit for performing the method. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method and kit for detecting endocrine disrupters. More specifically, the present invention relates to a method for detecting an endocrine disrupting substance which is an endocrine disrupter using aromatase activity as an index, and a kit therefor.

[0002]

2. Description of the Related Art Existing in the living environment of human beings, by acting similar to hormones such as sex steroid hormones of organisms including humans, or by interfering with the action of these hormones, the endocrine system of organisms is increased. Disturbing substances have been attracting attention. In recent years, reproductive abnormalities of wildlife caused by endocrine disrupting substances of this organism, such as atrophy of male penis and decrease in sperm count, maleification of conch females, etc., have been reported, and its effects on humans are worried. Has become a social issue (for example, "Challenge to environmental hormones", published by Nikkei BP:
See 1998 etc.).

Various hormones including sex steroid hormones are secreted by, for example, the testes and ovaries of living organisms, are carried into the body by the flow of blood and are present in the cytoplasm, nucleus, etc. Combined with
It is involved in the adaptation of living things to the environment, reproduction, production and storage of growth and developmental energy. When these hormones are affected by exogenous substances such as endocrine disruptors and fail to function normally, growth and reproduction of the organism are inhibited and the survival of the organism is compromised. In this way, endocrine disrupting substances are present in the living environment of living organisms and adversely affect the action of various hormones such as sex steroid hormones, and are therefore called “environmental hormones”. The environmental hormone is also an "exogenous endocrine disruptor" in Japan, and in overseas, Endocrine Disruptors:
EDs), Enviromental Endocrine Disruptors, Endocrine Disruptors
rupting Chemicals), Environmental Hormones (E
nviromental Hormones) is also called.

Typical examples of hormones affected by environmental hormones, which are substances that disturb the endocrine system of these organisms, include sex steroid hormones such as estrogen and testosterone, as well as insulin, thyroid hormone,
Examples include adrenaline and human growth hormone. At present, about 70 kinds of chemical substances are known as exogenous endocrine disrupting substances which are suspected to adversely affect the action of these hormones. Examples include toxic substances such as PCBs and dioxins; pesticides used as active ingredients for insecticides; substances used in the paint and dye fields such as bisphenol A, phthalates, and organic tins;
Examples include substances used as preservatives. Also,
It is suspected that there are substances having an environmental hormone-like action in natural products such as flavonoids contained in soybean. As described above, various substances in the environment surrounding the organism have a possibility of exerting an action of disturbing the endocrine system of the organism.

[0005] In particular, environmental hormones exert hormonal actions in extremely small amounts, and the adverse effects of environmental hormones on the fetuses, infants and other infants, in which various hormones play important roles, and the intake and accumulation of environmental hormones. There is concern about the adverse effects of the environmental hormones on the next generation of living organisms. Also, with regard to long-term environmental hormone research, each country has begun to take measures at the administrative and governmental level.

[0006] Thus, a technique for detecting how much an endocrine disrupting the endocrine system of an organism is present in blood and tissues, and the presence of the endocrine disrupter in the environment that adversely affects the ecosystem. There is an urgent need in the industry to develop technology for detecting and measuring quantity.

[0007] So far, the methods for detecting and evaluating the environmental hormones include a method in which a test substance is applied to an animal to see the effect, a method in which cultured cells are used to examine the binding of the test substance to hormone receptors such as estrogen, etc. Is being done.

However, these methods are disadvantageous in that they require complicated operation and long time. In addition, there are environmental hormones that cannot be detected by these methods, for example, they are present at extremely low concentrations below the standard that is said to be safe for the human body according to national standards, and they are not detected by known detection means. Considering that there is a substance that perturbs the endocrine system that is not recognized as an endocrine disrupter (a substance that acts on the hormone receptors of living organisms and exerts some influence on the endocrine system), it is conventionally known. It has been desired to establish an improved method that can detect and measure these substances more easily and with high sensitivity in place of the detection system.

More specifically, whether or not a substance (test substance) that affects the endocrine system of organisms including human beings, especially hormones, has a similar effect to that of hormones is, for example, human beings. It can be examined by detecting whether or not the test substance can bind to the hormone receptor possessed by cells, but human cells are required for this detection. However, it is generally difficult to obtain and prepare human cells. Potential human cells include cells derived from human cancer tissue, but establish such human cancer cell lines that can continue to maintain specific hormonal activity and can be easily increased in vitro. It can be expected to be quite difficult.

Therefore, in place of human cells, yeast cells into which a human hormone receptor gene and a transcription-coupling factor gene have been incorporated by a gene recombination technique to impart human hormone receptor expression ability have been utilized. Attempts have been made to examine the degree of binding between a test substance and a hormone receptor using an enzyme such as β-galactosidase that is simultaneously expressed as an index (see Japanese Patent Laid-Open No. 2001-299359 etc.). However, the yeast cells used in the method do not always reproduce the same state as the human metabolic system, and in fact, when the test substance is an environmental hormone that affects the endocrine system in a trace amount, However, the detection / measurement may not always be performed accurately.

Further, whether or not the test substance is an endocrine disrupter is detected by culturing a human breast cancer cell line into which a human-derived estrogen receptor gene has been introduced, in the presence of the test substance, and detecting a change in estrogen action. Or a method of detecting an endocrine disruptor by comparing the degree of binding of a test substance to the estrogen receptor with estrogen using Escherichia coli introduced with the human hormone receptor gene ( "Challenge to environmental hormones", published by Nikkei BP, 1998).

However, in all of the above-mentioned currently known environmental hormone measuring techniques, a chemical substance binds to a hormone receptor, and as a result, it promotes or inhibits the transcriptional activity of the responsible gene that occurs subsequently. It is a simple and highly sensitive measurement technology that can detect and measure chemical substances that affect the production amount of biohormones themselves by affecting the hormone synthase in the body. Is still under development.

[0013]

Therefore, the present invention provides an environment for finding a substance that affects the endocrine hormone synthesis system, instead of a technique for measuring the effect of a chemical substance on the endocrine system via the endocrine hormone receptor. The purpose is to provide a technique for measuring hormones.

[0014] In the process of earnestly researching to achieve the above object, the present inventors previously established cloned human ovarian granules established by long-term cell culture from tissues derived from human ovarian granule cell carcinoma patients. Membrane cell line KGN (Nishi, Y., et
We focused on the aromatase activity of al., Endocrinology, 142, 437-445 (2001)). As a result of further studies, the present inventors have found that the variation in aromatase activity of the cell line in the coexistence of a test substance serves as an indicator of the activity of environmental hormones, and the KGN cell line is functional. It is very similar to normal ovarian granulosa cells in that it has FSH receptor expression and relatively high aromatase activity,
From this, the more direct detection of environmental hormones of interest in mammalian systems by utilizing the KGN cell line.
It was found that the measurement system can be established.

More specifically, the present inventors utilize cells having an aromatase activity such as the above KGN cell line, and for various test substances, the aromatase activity of the cells measured in the presence thereof is When compared with the same value measured in the absence of the test substance, it was found that the obtained result well supports one hypothesis conventionally known for this kind of environmental hormone. The hypothesis is that organotin compounds such as tributyltin (TBT) and triphenyltin (TPT), which are the most studied series of many chemical substances among endocrine disruptors (environmental hormones) (mainly With respect to marine antifouling paints, its mechanism of action affecting the reproductive system of wildlife is associated with estrogen levels (ie reduced conversion of androgens to estrogen (eg aromatization)), It is regulated by increasing androgen levels (Bettin, C., et a
l., Helgolander Meeresunters, 50, 299-317 (1996):
Spooner, N., et al., Mar. Environ Res., 32, 37-49 (1
991)). That is, it was found that the aromatase activity of the above cell lines cultured in the presence of the test substance correlates with the action of the endocrine disrupter on the test substance, and the aromatase activity serves as an index of the endocrine disrupter action of the test substance.

From the above, the present inventors have succeeded in establishing a new detection and measurement system for environmental hormones using the fluctuation of aromatase activity of the above-mentioned specific cell line as an index, and to complete the present invention here. I arrived.

[0017]

The present invention provides a method and a kit for detecting an endocrine disrupter, which is an endocrine disrupter shown in items 1-12 below.

Item 1. A step of culturing a cell line having an aromatase activity in the presence or absence of a test substance under physiological conditions to measure the aromatase activity of the cultured cells, and the absence of the test substance Characterized in that it comprises a step of detecting a change in the aromatase activity of the cell line cultured in the presence of a test substance based on the aromatase activity of the cell line cultured below to identify a test substance that gives the change , A method for detecting a test substance as an endocrine disruptor using aromatase activity as an index.

Item 2. The detection method according to Item 1, wherein the cell line having aromatase activity is a human cell line having aromatase activity.

Item 3. The detection method according to Item 2, wherein the human cell line having aromatase activity is a human ovarian granulosa cell line.

Item 4. The human ovarian granulosa cell line is a KGN cell line
(RIKEN Gene Bank Deposit No .: RCB1154).

Item 5. Item wherein the aromatase activity is measured by any measuring method selected from [ ³ H] H ₂ O release assay method, ELISA method, RIA method and formic acid release assay method.
The detection method according to any one of 1-4.

Item 6. A detection kit for detecting endocrine disrupting substances using aromatase activity as an index, which comprises a cell line having aromatase activity and a reagent for measuring aromatase.

Item 7. The detection kit according to Item 6, wherein the cell line having aromatase activity is a human cell line having aromatase activity.

Item 8. The detection kit according to Item 7, wherein the cell line having aromatase activity is a human ovarian granulosa cell line.

Item 9. The human ovarian granulosa cell line is a KGN cell line
(RIKEN Gene Bank Deposit No .: RCB1154 No.) The detection kit according to Item 8.

Item 10. The aromatase activity is measured by any measuring method selected from [ ³ H] H ₂ O release assay method, ELISA method, RIA method and formic acid release assay method. The detection kit according to any one of 9.

Item 11. The reagent for measuring aromatase is [1β- ³
H] The detection kit according to any of items 6-10, which is androstenedione.

Item 12. The detection kit according to Item 11, wherein at least one member selected from the group consisting of dibutyryl / cyclic AMP, FSH, dexamethasone, menopasal, and gonadotropin is added to the aromatase measurement reagent.

[0030]

BEST MODE FOR CARRYING OUT THE INVENTION (1) Having aromatase activity
Cell line The cell line used for the detection (measurement) of the environmental hormone of the present invention is
There is no particular limitation as long as it is a cell line having aromatase activity, and examples thereof include cell lines obtained from human brain / neuronal cells and follicle cells. As one preferable representative example, human ovarian granulosa cell line KGN (RIKEN Gene Bank Deposit No .: RCB1154) can be exemplified.

The cell lines are not limited to those derived from humans, but those derived from other mammals such as rats, mice, monkeys, dogs, cats, sheep and rabbits, for example, granulosa cells of the follicles of these mammals. It may be a cell line having a stable aromatase activity established from the above. Further, as long as it has aromatase activity, it may be, for example, a cell line obtained from shellfish such as snails, oysters and mussels, and a cell line obtained from fish such as medaka, crucian carp and carp.

The KGN strain, which is one of the preferred cell lines, will be described in detail. The strain is an invasive ovarian granulosa cell carcinoma patient.
It was derived from (female) and the abnormal karyotype
Presented in 45, XX, 7q-, -22 (7q deletion and monozomy 22), with very low or no activity of 17β-hydroxylase and 17-lyase in the steroid synthesis pathway from cholesterol 4-Androsten
-3,17-dione is not synthesized. In addition, since the activity of 17β-hydroxysteroid dehydrogenase (17βHSDH) is sufficient, if 4-androstene-3,17-dione, estrone, etc. are added from the outside, testosterone, 17β and
-Estradiol (E) is synthesized. Further, the present cells have a relatively high aromatase activity, and the aromatase activity has characteristics similar to those of normal granulosa cells. The cell line is 4-androsten-
In the presence of 3,17-dione, estrone (E1) can be produced. In addition, since the cells can produce 17β estradiol from estrone (E1) by the activity of 17βHSDH, addition of 4-androstene-3,17-dione from the outside causes any activity of estrone and 17β estradiol. The strength of aromatase activity can also be evaluated by measuring. Moreover, if the testosterone produced by the addition of 4-androstene-3,17-dione can be specifically measured, the 17βHSDH activity in the steroid synthesis pathway can be measured. Also,
The cells show progesterone-producing ability by addition of HCG (human chorionic gonadotropin), and FSH (Follicle stimu
It is thought to express the lating hormone) receptor (Nishi, Y., et al., Endocrinology, 142 (No.1), 4
37-445 (2001), this KGN strain has been deposited with the RIKEN Gene Bank / Cell Development Bank of RIKEN under the RIKEN Gene Bank deposit number: RCB1154). (2) Test substance In the detection (measurement) method of the environmental hormone of the present invention, the test substance to be measured is already an environmental hormone (endocrine disruptor)
In addition to the chemical substances known as, those derivatives that are considered to exert endocrine disrupting activity and substances having endocrine disrupting activity that cannot be detected as an endocrine disrupter by the current technology are also included. Specific examples of substances currently known as environmental hormones include the followings.

Dioxin, polychlorinated biphenyls (PCB, Polychlorinated biphenyls), polybrominated biphenyls (PBB, Polybrominated biphenyls), hexachlorobenzene (HCB, Hexachlorobenzene), pentachlorophenol (PCP, Pentachlorophenol), 2, 4, 5-Trichlorophenoxy acetic aci
d, 2,4,5-T), 2,4-Dichlorophenoxyacetic acid (2,4-Dichloro
phenoxyacetic acid, 2,4-D), Amitrole,
3-Amino-1,2,4-triazole), Atrazine, Alachlor, Simazine, CAT, Hexachlorocyclohexane (HC
H), BHC), Ethyl parathion, Parathion
ion), carbaryl, NAC, chlordane (Chlo
rdane, Oxychlordane, trans-Nonachlor), 1,2-Dibrome-3-chloropropane, D
BCP), DDT (DDE and DDD), Kelthane, Dicofo
l), Aldrin, Endrin, Dieldrin, Methomyl, Endosulfan, Benzoepin, Heptachl
or), Heptachlor epoxid
e), Marathion, Methoxyc
hlor), Mirex, Nitrofen (Nitrofen,
NIP), Toxaphene, Tributyltin (Tri
butyltin, TBT) or triphenyltin (Triphenyltin;
Organotin compounds such as TPT),
Trifuralin, Nonylphenol
Alkyl phenol such as phenol or octylphenol, bisphenol A, di-2-ethylhexyl phthalate
(Di-2-ethylhexyl phthalate, DEHP), Butylbenzyl phthalate (BBP), Di-n phthalate
-Butyl (Di-n-butyl phthalate, DBP), Dicyclohexyl phthalate (DCHP), Diethyl phthalate (DEP), Benzo (α) pyrene, 2,4- Dichlorophenol (2,4-D
ichlorophenol), di-2-ethylhexyl adipate (Di (2
-ethylhexyl) adipate), benzophenone (Benzophenon
e), p-nitrotoluene (p-Nitrotoluene), 4-nitrotoluene (4-Nitrotoluene), octachlorostyrene (Octach
lorostyrene), aldicarb, benomyl (Ben
omyl), Kepone, Mancozeb, Maneb, Metiram, Metribuzin
buzine), Cypermethrin, Esfenvalerate, Fenvale
rate), permethrin, vinclozolin (Vi
nclozoline), Zineb, Ziram, Di-n-pentyl phthalate (DPP), Dihexyl phthalate (DHP), Dipropyl phthalate (DprP) , Styrene dimers and trimers, N-Butyl benzene, Estradiol
l), diethylhexyl adipate
te, DOA), trans-chlorlordane, cis-cholordane, p- (1,1,3,3-tetramethylbutyl) phenol (p- (1,1,3,3- Tetramethlbuty
l) phenol, TMBP), 2,4-dichlorophenoxyacetic acid (2,4-Di
chlorophenoxy) acetic acid, 2,4-PA), cadmium (C
Metals such as d), lead (Pb), mercury (Hg), etc.

Further, by utilizing as a test substance in the present invention,
In addition to the above, substances having endocrine disrupting effects that can be regarded as endocrine disrupters include, in addition to the above, eluates from industrial products, for example, decomposition products of main raw materials eluted by their use from plastic foods, plastic additives, plasticizers, etc. Is included.

The test substance in the present invention may be one that contains at least one of the above-mentioned environmental hormones or one that may possibly be present. Specific examples thereof include various agricultural chemicals, various industrial wastewaters, river water, Examples thereof include biological fluids such as blood. (3) Cultivation of cell line and measurement of aromatase activity In the assay and measurement method for environmental hormones of the present invention, first, the cell line is cultured to measure aromatase activity of the cultured cells. The culture of the cell line and the measurement of the aromatase activity can be performed according to various methods known in the art.

For example, culturing of a cell line is specifically 10% (v /
v) Using RPMI1640 medium with FCS, incubate 1 × 10 ⁴ cells / dish at 100% humidity and 5% CO ₂ at 37 ° C for 48 hours or more, and add 10% DSC to the medium before adding the test substance. DMEM / Ham's F-
12 It can be carried out by changing to a supplemented medium.

The aromatase activity is measured, for example, by measuring the radioactivity of [ ³ H] H ₂ O based on the conversion of [1β- ³ H] androstenedione (A) to estrone (E1) [ ³ H] H ₂ Can be performed by the O-free assay (Ackerman,
GE, et al., J. Clin. Endocrinology Metab., 53,
412-417 (1981)). The aromatase activity was measured by the method of Tanaka et al. (Tanaka, S., et al., Calcif.
Sue Int., 52, 107-109 (1993)). Furthermore, the aromatase activity can also be measured by a method such as an ELISA method, a RIA method, a formic acid release assay method.

The method for measuring the radioactivity of [ ³ H] H ₂ O will be described in detail. In the method, first, 10
% Fetal calf serum (FCS) (e.g. Hyclone Co., Ltd.) seeded in a petri dish containing a medium containing medium (for example, Falcon 3001 (Falcon) can be used), and then the culture medium,
Substituted with DMEM / Ham's F-12 containing 5.0-10% (V / V) DCS (dextran-coated and charcoal-treated FCS), 10 ^-5 to 10 ^-2 M (Bu) ₂ cAMP (Hereinafter, abbreviated as db-cAMP: manufactured by Wako Pure Chemical Industries, Ltd.) is added with the test substance or the test substance is added alone and incubated for 12 hours. Then, the test substance and 12.5 nM [1β- ³ H] androstenedione (NEN Life Science Product Co., Ltd .; 27.5 Ci / mmol) are further added and incubated for 6-12 hours. After the incubation, 1.0 ml of ice-cold 30% (w / v) trichloroacetic acid (TCA) is added to the medium (2.0 ml), and the mixture is stirred and centrifuged for protein removal. Protein concentration is determined after harvesting cells using 0.25% (w / v) trypsin-1.0 mM EDTA. To measure the amount of [ ³ H] H ₂ O released,
The medium was extracted and the method of Tanaka et al. (Tanaka, S., et al., C
alcif. Tissue Int., 52, 107-109 (1993)), collect the medium supernatant, extract with 5 ml of chloroform (after adding chloroform to the collected solution, stir, centrifuge, and collect 2 cc of chloroform). Add 2cc of 5% charcoal-0.5% dextran, stir, let stand for 15 minutes, then spin at 3000 rpm for another 15
Centrifuge for minutes. 1.8 cc of the supernatant thus obtained is placed in a scintillation vial, 10 ml of scintisol is added, and the radioactivity of [ ³ H] H ₂ O is measured.

The amount of radioactivity of [ ³ H] H ₂ O is the value of blank from each test sample (a non-specific value which is counted by adhesion of a radioactive substance to a tube when the same operation is performed without adding cells). Correct count by blanking). Thus, the aromatase activity of the cultured cells before and after the test substance administration can be measured. In addition, the amount of protein in the cells was 0.1N NaOH.
Micro BCA kit (Pierce Chemical C
o., company) can be used for measurement. Thus, the aromatase activity of the cultured cells before and after administration of the test substance can be measured as pmol / mg cellular protein.

Although KGN cells do not normally produce estrone or estradiol, when androstenedione or testosterone which is a substrate for aromatase is added, aromatase in KGN cells produces estradiol from estrone or testosterone from androstenedione. create. Therefore, the aromatase activity of the KGN cells can also be determined by measuring the amount of estrone or estradiol produced by the KGN cells as an index. Hereinafter, this method will be described in detail.

That is, after culturing the cultured cells in a culture medium having 10% FCS, the culture medium was replaced with DMEM / Ham's F-12 containing 5% (V / V) DCS and cultured for 24 hours, afterwards,
Various concentrations of the test substance (usually 10 ⁻¹² M to 10 ⁻⁶ M) are added to the medium and incubated for a further 48 hours. After incubation, 10 ⁻⁶ M 4-androstene-3,17-dione is added to the cells. The culture is further continued for 12 to 48 hours, the culture medium is collected, and the amount of estrone or estradiol secreted in the culture medium is measured using a commercially available or ELISA or RIA. in this case,
Aromatase activity was measured as estrone or estradiol in a solvent control containing no test substance (reference value, 100%) by comparing the measured value of estrone or estradiol in the cell culture system containing the test substance with the above reference value. Can be calculated as the relative value. In this case, taking the concentration of the test substance on the horizontal axis and taking the estrone amount as an index of the aromatase activity on the vertical axis, the relative value (%) of the aromatase activity at each concentration of the test substance is easily obtained. be able to.

Furthermore, the aromatase activity of KGN cells is
It can also be detected and measured by the formic acid release assay method.

In this formic acid release assay method, one molecule of formic acid (formic acid) is converted when one molecule of androstenedione having 19 carbon atoms is converted to estrone having 18 carbon atoms by aromatase (p-450arom) in the above-mentioned reaction. HCOOH) is generated, and formic acid in this culture solution is used as an index for aromatase activity. The measurement (quantification) of formic acid in the culture solution can be performed by, for example, capillary electrophoresis, HPLC, ion chromatography, gas chromatography and the like.

More specifically, after culturing the cultured cells in a culture medium containing 10% FCS, the medium is added to DMEM / Ham 'containing 5% DSC.
Replace with sF-12 and incubate for 24 hours. At this time, if necessary, (Bu) ₂ cAMP can be added to enhance the aromatase activity. Then, various concentrations of test substance (usually 1
0 ^-12 M to 10 ^-6 M) is added to the cells and incubated for 12 to 48 hours. After the incubation, a constant amount (usually 10 ⁻⁶ M) of 4-androstene-3,17-dione is added and the culture is continued for further 12 to 48 hours. Collect the culture medium,
Formic acid (HCOOH) secreted in the culture medium is quantified by capillary electrophoresis, HPLC, ion chromatography, gas chromatography and the like. The aromatase activity is measured as a relative activity amount with respect to a solvent control containing no test substance as 100%. In the method, for example, the concentration of the test substance is plotted on the horizontal axis. Alternatively, it is plotted as aromatase activity on the vertical axis as% of formic acid production. (4) Detection of changes in aromatase activity and
Detection, measurement of environmental hormones of the present invention, the assay method, the cells are cultured under physiological conditions in the presence and absence of a test substance, the aromatase activity of each cultured cell is measured, the test substance Based on the measured value in the absence of the test substance, whether or not the measured value in the presence of the test substance fluctuates in comparison with the above reference value, and whether it is rising or falling It specifies the test substance.

In the hormone synthesis system in the living body, estradiol, which is a female hormone, is synthesized from cholesterol taken in as a food through several enzymatic reactions. Aromatase plays an important role in synthesizing C18 hormones such as estradiol from C19 hormones such as testosterone. When this hormone synthase is inhibited, estradiol is no longer synthesized, so substances with such properties have endocrine disrupting effects. For example, TPT, which is known to generate a penis in female snails, has an aromatase inhibitory activity, and thus is attracting attention as a new endocrine disrupting substance. In addition, if a chemical substance whose activity affects living organisms is unknown, it affects the aromatase activity by affecting the live hormone synthesizing system, whether it decreases or increases the activity. It will disrupt the endocrine system. The method of the present invention provides an effective means for screening such endocrine disrupting substances. (5) Detection and measurement kit of the present invention The present invention further provides a cell line having aromatase activity and a component for detecting fluctuations in aromatase activity.
Provided is a detection kit for detecting endocrine disrupting substances, which comprises (reagents and the like), using aromatase activity as an index.

The cell line as an essential component of the kit is as described above. The amount contained in the kit of these cell lines is not particularly limited and can be appropriately determined according to the type and concentration of the test substance to be detected and measured. Generally, the test substance concentration is 0.1 nM-1000 nM / m
For l, preferably 1 nM-200 nM / ml, 1 × 10 ⁴ -1 × 10 ⁶ cells / well or dish can be used.

The component contained in the kit for detecting and measuring the test substance using the aromatase activity of the cells as an index and the amount contained in the kit are, for example, 12.5 nM [1β- ³ H] andro. Stenetion, 10 ^-4 M-10 ^-2 M db-cAMP, 50-500 ng / ml FSH, 10 ^-8 M-
DMEM / containing 10 ^-5 M dexamethasone or 50-5000 mIU / ml menopausal gonadotropin (MG) and 5.0% (v / v) DCS
A preferable medium for culturing mammalian cells having aromatase activity such as Ham's F-12 1: 1 mixture can be exemplified. Specifically, DMEM / Ham's containing 5% (v / v) DSC as a medium, such as androsterone as a substrate, testosterone, etc.
F-12 etc. can be mentioned.

The above-mentioned component FSH, dexamethasone or MG is used for stimulating the aromatase activity of a cell line, and for the purpose of increasing the measurement sensitivity of the kit or the above-mentioned assay method for environmental hormones of the present invention, db-c
It can be exemplified as a component having an activity similar to that of AMP.

The above-mentioned kit is effective for detecting and measuring endocrine disrupting substances as environmental hormones, and therefore can be used as a screening kit for endocrine disrupting substances.

[0050]

INDUSTRIAL APPLICABILITY According to the present invention, the effect of the test substance on the endocrine system of an organism is determined in a more realistic environment in the amount that the environmental hormone (endocrine disruptor) as a test substance actually affects the organism. Can be detected by a system that reflects a state close to
It is possible to provide a technique for measuring an endocrine disrupter using a change in aromatase activity as an index.

According to the present invention, a test substance is added to a cell line having aromatase activity, and a decrease or increase in aromatase activity due to the test substance is detected to detect a test substance having an aromatase activity decreasing action or increasing action. It is possible to provide a method for measuring an endocrine disruptor which screens for an endocrine disruptor.

Further, according to the present invention, it is possible to provide a kit using the above-mentioned method for measuring environmental hormones.

[0053]

EXAMPLES Examples will be given below to describe the present invention in more detail, but the present invention is not limited to the following examples.

The materials and reagents used in the following examples are as follows.

Tributyltin (TBT) and triphenyltin (TPT) are commercially available from Sigma Chemical Company (Sigm
a Chemical Co.). Both compounds are dissolved in ethanol and the final concentration of ethanol in the cell growth medium is 0.1% (v / v).

YM511, a specific aromatase inhibitor, was produced by Yamanouchi Pharmaceutical Co., Ltd. (Kudoh, M., et al., J. Steroid. Bioche
m. Mol., 54, 265-271 (1995)).

DMEM / F12 and fetal calf serum (FCS) were purchased from Gibco BRL.

[1β- ³ H] Androstenedione (androsten
edione) was purchased from Amersham Pharmacia Biotech.

The Taq DNA polymerase, pGEM-T easy vector, pGL3 luciferase reporter vector and Dual-luciferase reporter assay system manufactured by Promega were used.

The plasmid DNA isolation kit and SuperFect transfection reagent were purchased from Qiagen.

All the primers for PCR are known, and were synthesized by requesting Amersham Pharmacia Biotech. The base sequence of the used primer is SEQ ID NO:
Shown in 1 and: 2.

Dibutyryl cAMP (db-cAMP) was purchased from Sigma Chemical Co.

In each of the following examples, the aromatase activity measurement value, E2 value and 450 arom for control cells were measured.
Variations in activity in cells treated with mRNA and drug were statistically analyzed by Student's t-test. A p <0.05 was considered statistically significant. Furthermore, all data in this example are shown as mean ± SD.

[0064]

Example 1 Cell and Cell Growth Assay In this example, a human ovarian granule cell line
losa-like tumor cell line; KGN cell line, RIKEN Gene Bank deposit number: RCB1154) was used. The cell line at 37 ° C.
Maintained in DMEM / F12 medium containing 10% FCS in air with 5% CO ₂ . a) Effect of test substance on cell proliferation For cell proliferation test, KGN cell line was added to DMEM / F containing 10% FCS.
1x10 ⁴ cells / well in a 24-well plate containing 12 media
The wells were seeded in an amount. Then, an ethanol solution of TBT or TPT as a test substance (final concentration:
0.1% (v / v)) of 2 ng / ml, 20 ng / ml or 1000 ng / ml was added to each well to culture the cells (experimental cell group).

On the other hand, a control cell group was prepared in which the KGN cell line was treated only with ethanol in the same manner except that TBT and TPT were used in the above.

The culture medium containing the test substance or ethanol in each group was replaced every two days.

After further washing, the cells in the wells were treated with trypsin, and the number of viable cells in each well was counted every 48 hours for 7 days using a hemocytometer. The number of viable cells was evaluated by the trypan blue exclusion method.

The results are as shown below.

(1) 20 ng / ml or less concentration (2 ng / m
It was revealed that the presence of TBT or TPT in l) did not affect the cell proliferation of KGN cells.

(2) TBT or TPT at a concentration of 1000 ng / ml or more
Was found to be highly toxic to KGN cells (all cells died within 24 hours).

From the above, it was reported to cause imposex of marine organisms (Bryam, GW, et al., J. M.
ar. Biol. Assoc. UK 66, 611-640 (1986): Gibbs, P.
E., and Bryan, GW, J. Mar. Biol. Assoc. UK 66, 7
67-777 (1986): Horiguchi, T., et al., Thaisclaviger
Environ. Pollut., 95, 85-91 (1997)) 20 ng / ml TBT
Alternatively, since the concentration of TPT was found to be a concentration that does not substantially affect the growth of KGN cells, the concentration of TPT for detecting the endocrine disrupting effect of the present invention will be described below. The test was conducted.

[0072]

Example 2 Aromatase Activity Test Aromatase activity was reported by Mu et al. (Mu, YM, et al.
al., BBRC, 271 (3), 710-713 (2000)) and [ ³ H] H ₂ O based on the conversion of [1β- ³ H] androstenedione (A) to estrone (E1). Was determined by measuring the radioactivity of the.

Prior to the experiment, KGN cells were incubated with 5% DC for 48 hours.
Contains S (FCS coated with dextran and charcoal treated)
Cultured in DMEM / F12.

After treatment of KGN cells (1 × 10 ⁴ cells / well) with 2 ng / ml of either TBT or TPT, 12.5 nM
[1β- ³ H] Androstenedione was added and the cells were then further cultured for 6 hours.

After culturing, the medium (2.0 ml) was collected, 1 ml of ice-cold 30% trichloroacetic acid was added, and the mixture was stirred and centrifuged to remove proteins. Then, collect the collected medium supernatant for 5 m.
It was extracted with l chloroform and the extract was centrifuged to remove chloroform.

Further, 2 ml of 5% charcoal / 0.5% dextran was mixed with the aqueous supernatant and incubated for 30 minutes. Then the mixture was centrifuged at 3000 rpm for 15 minutes. Then, 1.8 ml of the supernatant was collected in a scintillation vial, 10 ml of scintisol was added, and [ ³ H] H ₂ O radioactivity was measured.

The measured radioactivity of [ ³ H] H ₂ O was measured using a micro BCA kit (Pierece Chemical Co.).
Normalized based on the determined protein concentration and expressed as pmol / mg protein / 6 hours.

As a result, it was revealed that TBT or TPT suppressed the aromatase activity of KGN cells.

That is, in order to identify the regulation of aromatase activity of KGN cells, first, 20 ng / ml of TBT was added in the presence or absence of db-cAMP for 48 to 72 hours.
Alternatively, TPT and cells were incubated together. Aromatase activity was then assessed by [ ³ H] H ₂ O release assay.

The basal level of aromatase activity in KGN cells was 0.435 ± 0.041 pmol / mg protein / 6 hours.

Addition of 20 ng / ml of TBT or TPT, respectively,
Aromatase activity was suppressed to about 30% of the above basal level (P <0.05) (Basal (Control) and db-cAMP (Control) in FIG. 1A).
reference).

[0082] Figure 1A is for indicating the modulation of aromatase activity in cultured KGN cells, 10 ^{- 4} the presence of db-cAMP in the M (display in the drawing as "db-cAMP") or absence (figure "B
labeled asal)) for 48 h to KGN cells in TBT or TPT 2
It is the graph which calculated | required the aromatase activity of the cultured cell treated by 0 ng / ml, or a non-treated cultured cell (indicated as "Control" in the figure). In the figure, the horizontal axis represents the cultured cells under each condition, and the vertical axis represents the aromatase activity (pm
ol / mg protein / 6hr). In the figure, the white bar graph is the control result, and the shaded bar graph is TB.
It is a result when it processed by T20ng / ml, and the bar graph shaded with a dot is a result when it processed by TPT20ng / ml.

On the other hand, treatment of 10 ⁻⁴ M db-cAMP caused a 1.7-fold increase in aromatase activity above the basal level (see graphs of Basal and db-cAMP controls in FIG. 1A). This increase was also suppressed by 20 ng / ml TBT or TPT (see graph of db-cAMP TBT and TPT in Figure 1A).

To see the effect of 20 ng / ml TBT on basal or db-cAMP stimulated aromatase activity, we tested over a 7 day time course. The results are shown in Figure 1B.

In FIG. 1B, the horizontal axis shows the elapsed days (days), and TB
Aromatase activity of cultured KGN cells treated with T20ng / ml (without addition of db-cAMP) (displayed as open bar graph in the figure)
And aromatase activity of cultured KGN cells treated with 20 ng / ml of TBT in the presence of 10 ⁻⁴ M db-cAMP (displayed as a shaded bar graph in the figure) (the vertical axis is The percentage (%) with respect to the control is shown).

As is evident from FIG. 1B, a significant inhibition of aromatase activity was the earliest after 3 drug addition, either basal or stimulated by 10 ⁻⁴ M db-cAMP. Observed for days, and maximal inhibition was observed on day 7.

Then E2 in db-cAMP stimulated cells treated or not treated with TBT or TPT for 7 days
Production was evaluated by a similar test.

The results are shown in FIG. 1C. In FIG.1C, the horizontal axis represents control, cells cultured by adding 2 ng / ml of TBT or TPT to the cell medium and cells cultured by adding 20 ng / ml of the same substance, and the vertical axis represents androstenedione relative to control or The relative production ratio (%) of estradiol is shown. In the figure, the open bar graph is the result of control, the shaded bar graph is the result of TBT-treated cells, and the dotted bar graph is TPT.
This is the result in the treated cells.

From the results shown in FIG. 1C, 20 ng / ml for 7 days
Treatment with TBT or TPT was consistent with the observed changes in aromatase activity (see results on day 7 of Figure 1B), causing a significant reduction in E2 concentration in the medium.

[0090]

Example 3 Estradiol Content Analyzed by RIA a) To demonstrate that the measured aromatase activity truly reflects its ability to produce estrogen, cells were treated for 7 days with various concentrations of TBT or Process with or without TPT,
Then 12 hours, 4-androstene-3,17-dione (1 μM)
Was further added and further incubated. The medium was collected and the E2 (estradiol) content in the medium was determined by specific RIA.
(Manufactured by SRL).

B) RNA extraction and RT-PCR analysis RNA extraction and RT-PCR analysis for aromatase mRNA was performed according to Ikuyama et al. (Ikuyama, S., et al., Clin. Endocri).
Nol., 48 (5) 647-654 (1998)).

Thus, in order to investigate whether changes in aromatase activity of KGN cells were correlated with comparable changes in the level of p450arom mRNA, whole KGN cells were treated with 20 ng / ml TBT non-treated for 48 hours. CCD image sensor, Density
itograph AE6900F, manufactured by Atto) was used to quantify the staining property of ethidium bromide.

The results are shown in FIG. 2 (vertical axis: relative aromatase mRNA level, horizontal axis: control and TBT-treated KGN cells), and it was found that TBT decreased the expression level of p450arom mRNA. .

Changes in p450arom mRNA levels correlated with comparable changes in aromatase activity. Thus, reduced aromatase activity in TBT-treated KGN cells correlated with reduced p450arom mRNA levels.

C) Plasmid structure and luciferase analysis Decreased expression of P450arom mRNA results in ovarian granulosa cells (Nishi, (2001): Means, GD, et al., Mol. Endocrin.
ol., 5, 2005-2013 (1991): Carlone, DL, Richards,
JS, et al., Mol. Endocrinol., 11, 292-304 (1997))
To further identify whether it is regulated through promoter II, the major promoter of Escherichia coli, the luciferase activity was determined by pGL3 basic vector (Mu, Y., et al., Mo.
l. Cel. Endocrinol., 181, 239-248 (2001)) 1Kb P45
It was identified using the 0arom promoter II expression construct.

Transfection was carried out using Superfect Reagent according to the manufacturer's instructions.

Briefly, KGN cells (1 × 10 ⁵ ) were seeded in 6-well plates 12 hours prior to transfection and they were transfected with 2 mg DNA.

As an internal standard, 5 ng of Renilla luciferase control reporter pRL-CMV was added to each well to assess transfection efficiency.

On the day of transfection, KGN cells were treated with or without TPT or TBT.
KGN cells were maintained at 37 ° C for 48 hours, after which they were lysed and harvested. It was then subjected to luciferase analysis using the Dual-luciferase reporter assay system according to the manufacturer's instructions.

As a result, the promoter activity was identified and shown as a fold increase in neutralized luciferase activity relative to Renilla luciferase activity (called relative luciferase activity).

The luciferase reporter driven by the p450arom promoter II is an empty vector pGL3-
It displayed 100 times higher luciferase activity compared to basic.

The results are shown in FIG.

FIG. 3-A shows KGN transfected with a fusion gene construct containing the 1-kb P450arom promoter II.
Cells were treated with 20 ng / m 2 in the presence or absence of 10 ^-4 M cAMP.
It is the graph which calculated | required the relative luciferase activity after 48-hour culture | cultivation in the culture medium which added 1 TBT or TPT. In Fig. 3-A, the open bar graph shows the control, and the bar graph shaded with diagonal lines is KGN cultured in the medium containing 20 ng / ml TBT.
Shows cell results, dot-shaded bar graph is 20ng
The results of KGN cells cultured in a medium containing / ml TPT are shown.

FIG. 3-B shows that KGN cells transfected with the fusion gene construct containing 1-kb P450arom promoter II and bovine Ad4B expression vector were treated with or without 10 ⁻⁴ M cAMP. 2 is a graph showing the relative luciferase activity obtained after 48 hours of culture in a medium supplemented with 20 ng / ml TBT or TPT. In the figure, an open bar graph shows the results of the original KGN cells (cells not transfected at all), and a bar graph shaded with a horizontal line shows control cells (transfected but TBT, TPT).
(Without added), the shaded bar graph shows the results of KGN cells cultured in 20 ng / ml TBT-added medium, and the dot shaded bar graph shows the KGN cells cultured in 20 ng / ml TPT-added medium. The result is shown.

From the results shown in FIG. 3, 10 ⁻⁴ M of db-cAMP was obtained.
Caused a 1.7-fold activation of the promoter (Fig. 3-
(See contrast between Basal control of A and db-cAMP control).

Thus, luciferase activity was reduced by the addition of TBT in both the basal state (Basal) and the state stimulated by cAMP (see FIG. 3-A). In addition, forced expression of Ad4BP / SF-1
8-fold activation (null expression vector in Figure 3-B)
(See contrast with control).

The Ad4BP / SF-1 dependent increase in luciferase activity was significantly suppressed by both TBT and TPT.

[0108]

[Example 4] Effect of environmental hormones on aromatase activity of ovarian granulosa cell KGN cells In the same manner as in Example 2, TBT, TPT, benomyl (Benomy)
l), Heptachlor, Benzopyrene
The same test was repeated using each compound shown in FIGS. 4 and 5 such as rene) as a test substance.

The results are shown in FIGS. 4 and 5.

In FIGS. 4 and 5, the horizontal axis represents KGN when each test substance was added, based on the case where no test substance was added.
The relative aromatase activity (%) of the cells is shown, and the vertical axis shows each test substance.

Each compound as a test substance shown in FIGS. 4 and 5 is represented by the following name or abbreviation. P-nitrotoluene; P-nitrotoluene pentachlorophenol; Pentachlorophenol α-benzopine; a-benzopin 2,4-diclophenoxyacetic acid; 2,4-DPC benzophenone; Benzophenone γ-hexachlorocyclohexane; g-BHC β-benzopine B-benzopine benzo (α) Pyrene; Benzo (a) pyrene Alacrole; Alachlor β-Benzenehexachloride; b-BHC 2,4,5-Trichlorophenoxyacetic acid; 2,4,5-T Nitrophene; NIP Di-2-ethylhexyl phthalate DEHP Nonylphenol; Nonylphenol Atrazine; Atrazine Di-n-butyl phthalate; DBP Vinclozoline; Vinclozoline Kelsen; Kelthane diethyl phthalate; DEP Dicyclohexyl phthalate; DCHP Bisphenol A; Bisphenol A Metribuzin; Metribuzine Methoxychlorimazine; n-butylbenzene 1,2-dibromo-3-chloro Propane; DBCP diethylhexyl adipate; DOA malathion; Marathion cyperthrin; Cypermethrin fenvalerate; Fenvaleraye), trifluralin; Trifuralin amitrole; Amitrole mesomil; Methhomyl carbaryl; Octylphenol p- (1,1,3,3-Tetramethylbutyl) phenol; TMBP Octachlorostyrene; Octachlorostyrene Benomyl; Benomyl Hexachlorobenzene; Hexachlorobenzene Esfenvalerate Tributyltin; tributylin triphenyltin; triphenyltin dichlorodiphenyltrichloroethane; DDT DDT Metabolites of DDD; metabolites of DDT (1,1 '-(dichloroethenylidene) bis [4-chlorobenzene]); DDE endrin; Endrin heptachlor; Heptachlor transnonachlor; Trans-nonachlor transchlordane; Trans-ch olordane cis-cholordane; oxy-chlorlordane Oxychlordane Aldrin; Aldrin Dieldrin.

From the results shown in FIGS. 4 and 5, benomyl, heptacol, and benzopyrene, which are known as endocrine disrupters, surprisingly increased the aromatase activity of KGN cells (relative aromatase activity exceeded 100%). ). In particular, this study revealed that benomyl, which is known as a pesticide, increases aromatase activity about 3-fold. Although benomyl was known to be capable of inducing male infertility, the results of this test support the hypothesis that the relative estrogen excess due to increased aromatase activity is the cause.

[0113]

[Sequence list]                                SEQUENCE LISTING <110> Ostuka Pharmaceutical Co., ltd., Shin NAWADA and Toshihiko YANASE <120> A ditection method for endocrine disruptors <130> 4102JP <160> 2 <170> PatentIn Ver. 2.1 <210> 1 <211> 20 <212> DNA <213> Artificial <223> primer sequence (sense) <400> 1 cggccttgtt cgtatggtca 20 <210> 2 <211> 20 <212> DNA <213> Artificial <223> primer sequence (antisense) <400> 2 gtctcatctg ggtgcaagga 20

[Brief description of drawings]

[FIG. 1A] FIG. 1A shows that 10 ⁻⁴ M dibutyl cyclic AMP (d
FIG. 3 is a graph showing the effect of TBT and TPT on the aromatase activity of cultured KGN cells in the presence or absence of (b-cAMP).

FIG. 1B is a graph showing the effect of 20 ng / ml TBT on aromatase activity in the presence or absence of 10 ⁻⁴ M db-cAMP for 7 days or longer.

FIG. 1C is a graph showing the effect of TBT and TPT on estradiol (E2) production in cultured KGN cells.

FIG. 2 is a graph showing the effect of TBT on P450 arommRNA expression relative to that of β-actin in cultured KGN cells.

FIG. 3-A shows luciferase activity regulated by promoter II of P450 arom in KGN cells.
FIG. 3-B is a graph showing the effect of TBT and TPT, and P450 when co-transformed with fetal bovine Ad4BP expression vector (RSV / Ad4BP) or an empty expression vector as a negative control into KGN cells. TB for luciferase activity regulated by arom promoter II
It is a graph which shows the influence of T and TPT.

FIG. 4 is a graph showing the effect of various compounds on the relative aromatase activity (% of control) in KGN cells cultured using the same method as in Example 3.

FIG. 5 is a graph showing the effect of various compounds on the relative aromatase activity (% of control) in KGN cells cultured using the same method as in Example 3.

[Procedure amendment]

[Document name] Consignment number change notification

[Submission date] October 2, 2002

[Former name of depositary institution] RIKEN Biosource Center (RIKEN Gene Bank / Cell Development Bank)

[Old trust number] RCB1154

[Name of new depositary institution] National Institute of Advanced Industrial Science and Technology Patent Microorganisms Depositary Center

[New contract number] FERM BP-8171

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) (C12Q 1/02 C12R 1:91 C12R 1:91) C12N 15/00 A (C12Q 1/25 C12R 1: 91) (72) Inventor Shin Nawada, 3-1-1, Madade, Kyushu University, Higashi-ku, Fukuoka-shi, Fukuoka (72) Inventor, Toshihiko Yanase, 3-1-1, Madade, Kyushu University, Higashi-ku, Fukuoka, Fukuoka ( 72) Inventor Mitsuru Iida 232-1 F-term, Iwajira, Naraya-machi, Naruto-shi, Tokushima Prefecture (reference) 4B024 AA11 CA05 HA14 4B063 QA01 QQ40 QR77 QS35 QX07

Claims (12)

[Claims] 1. A step of culturing a cell line having an aromatase activity in the presence or absence of a test substance under physiological conditions to measure the aromatase activity of the cultured cells, and in the absence of the test substance. Characterized in that it comprises a step of detecting a change in aromatase activity of the cell line cultured in the presence of a test substance on the basis of aromatase activity of the cell line cultured in, and specifying a test substance that gives the change. A method for detecting a test substance as an endocrine disrupting substance using aromatase activity as an index. 2. The detection method according to claim 1, wherein the cell line having aromatase activity is a human cell line having aromatase activity. 3. A human cell line having aromatase activity,
The detection method according to claim 2, which is a human ovarian granulosa cell line. 4. The detection method according to claim 3, wherein the human ovarian granulosa cell line is a KGN cell line. 5. The aromatase activity is measured by any measurement method selected from [ ³ H] H ₂ O release assay method, ELISA method, RIA method and formic acid release assay method.
The detection method according to any one of 4. 6. A detection kit for detecting an endocrine disrupter using aromatase activity as an index, which comprises a cell line having aromatase activity and a reagent for measuring aromatase. 7. The detection kit according to claim 6, wherein the cell line having aromatase activity is a human cell line having aromatase activity. 8. The detection kit according to claim 7, wherein the cell line having aromatase activity is a human ovarian granulosa cell line. 9. The detection kit according to claim 8, wherein the human ovarian granulosa cell line is a KGN cell line. 10. The aromatase activity is measured by any measurement method selected from [ ³ H] H ₂ O release assay method, ELISA method, RIA method and formic acid release assay method. The detection kit according to any one of 9. 11. The detection kit according to claim 6, wherein the reagent for measuring aromatase is [1β- ³ H] androstenedione. 12. The detection kit according to claim 11, further comprising at least one selected from the group consisting of dibutyryl cyclic AMP, FSH, dexamethasone, menopasal, and gonadotropin, in addition to the reagent for measuring aromatase.