KR100789726B1 - Methods for detection and toxicity evaluation of environmental toxic substances using amphibian ovarian follicles - Google Patents

Abstract

A method for detecting an environmental toxic substance and evaluating the toxicity thereof is provided to objectively and scientifically detect the substance influencing on propagation of animals simply, economically and ultimately by using amphibian ovarian follicles, thereby providing basic materials capable of determining the influence made by the substance on human health. A method for detecting an environmental toxic substance or evaluating the toxicity of the environmental toxic substance comprises the steps of:(a) culturing amphibian ovarian follicles in the presence of a steroid generation promoting agent and a candidate material; and (b) quantifying the steroid generated from the follicles to determine whether the candidate material is an environmental toxic substance or to evaluate the toxicity of the environmental toxic substance. A kit used for the method comprises amphibian ovarian follicles, a culture medium for culturing the amphibian ovarian follicles, and a quantifying tool of steroid generated from the amphibian ovarian follicles.

Description

Methods for detection and toxicity evaluation of environmental toxic substances using amphibian ovarian follicles}

1 is a diagram showing a model for detecting and toxicological evaluation of environmentally harmful substances using steroid synthase as an indicator;

2 is a diagram schematically showing a method for detecting environmentally harmful substances using a culture system of amphibian follicles;

3 is a diagram schematically illustrating a reversible or irreversible determination method of environmentally harmful substances toxicity using a culture system of amphibian follicles;

4 is a diagram schematically showing a method for identifying steroid-producing enzymes acting on environmentally harmful substances using the culture system of amphibian follicles;

5 is a graph showing the inhibitory effect of P450 _scc enzyme activity of organotin, heavy metals and imidazole series antifungals;

6 is a graph showing the results of reversible or irreversible determination of organotin, heavy metal and imidazole series antifungal toxicity;

7 is a graph showing the results of identification of steroid synthase with organotin;

Figure 8 is a graph showing the results of identification of steroid synthase acting imidazole series antifungal agent;

9 is a graph showing the results of identification of steroid synthase with heavy metals.

The present invention relates to a method for detecting environmental toxicity using amphibian ovarian follicles and to evaluate toxicity, and more particularly, to culture amphibian follicles in the presence of steroid production promoters and candidates, Quantitatively determining whether a candidate is an environmentally hazardous substance and / or evaluating its toxicity.

Regulation of reproductive activity in vertebrates consists of the interaction of hormones produced in the hypothalamus-pituitary-gonad. In particular, steroid hormones produced in the gonads not only directly affect the formation of spouses, but also affect the development and maintenance of secondary sexual characteristics, reproductive activity, and the like. Many of the chemicals produced by the industrial activities of modern humans are known to inhibit or interfere with the normal hormonal activity of humans and wildlife. The US Environmental Protection Agency (EPA) designates extracorporeal substances that interfere with the production, release, transport, metabolism, binding, action, or excretion of hormones in the body as endocrine disruptors. Endocrine obstructions known to date are pesticides, persistent organic halogen chemicals, plastic-related substances, heavy metals, etc., because they do not easily decompose in the natural environment or in vivo, and may accumulate through the food chain. It has the property of prolonging and widening. The US Environmental Protection Agency, together with the Toxic and Disease Registrar (ATSDR), presents the CERCLA Priority List every two years since 1997, taking into account human exposure frequency, toxicity, and potential. have. Among the 475 assessments conducted so far, the top 275 major hazardous substances were heavy metals (arsenic (As), lead (Pb), mercury (Hg) and cadmium (Cd)) and endocrine disruptors (PCBs, PHAs, benzo (A) Pyrene) was always included. Therefore, there is an urgent need to develop various indicators and methods for evaluating these substances and their toxicity.

In response to these demands, research on endocrine disruptors and heavy metals in developed countries has focused on developing assessment models and methods for identifying toxicity, identifying mechanisms of action, and identifying risks. In recent years, the detection and toxicity assessment of harmful substances mainly use biochemical and molecular biological markers, which not only can detect harmful substances much more quickly and accurately than studies at the individual level, but also reveal the mechanism of action. This is because it can be interpreted into individual level and ecological environment. For this reason, developed countries use steroid synthase and its genes, steroid receptors and their target genes most often as indicators of toxicity evaluation. Among the steroid-producing enzymes, P450 _scc , 3β-HSD (hydroxysteroid dehydrogenase), and P450 _arom activity and gene expression are used as indicators to evaluate the toxicity of various harmful substances such as endocrine disruptors and pesticides. This is because these are enzymes that act as regulators or lead the production of estrogens during the production of steroids (see Figure 1).

Toxicological assessment of environmentally harmful substances, including endocrine disruptors, ultimately requires the use of biological assays. Most bioassays are at the level of cell culture, but they are expensive to maintain and culture. There was a problem falling.

On the other hand, there are many studies on the toxicity evaluation of harmful substances in Korea, but mainly on the harmful substances themselves, such as quantitative analysis of substances accumulated in the eco-environment, animals and the human body. Although studies on endocrine obstructions in Korea are relatively more conducted than other harmful substances, the amount of research is relatively small compared to the types and amounts of substances produced and consumed. In addition, research tends to cover the presence or absence of reproductive toxicity, and research on the target cells and mechanism of action of the substance is very limited. This is because the detection of endocrine disruptors in Korea and its toxicity evaluation have been mainly dependent on in vitro tests (E-screen test, ER competitive binding assay, etc.), which are methods of detection and self-toxicity evaluation. Therefore, the method used in Korea can be usefully used for the detection of xenoestrogen, but the detection of substances whose harmful substances do not mediate hormone receptors, orandrogen and progestin characteristics. Reveals a very vulnerable part. As such, only a few studies developed in advanced foreign countries have been conducted to identify and detect the toxicity of the substance itself. However, there has been no development of objective indicators or toxicity assessment techniques suitable for our situation. Therefore, the development of objective biochemical and molecular biological indicators capable of quickly and accurately detecting substances requiring toxicity evaluation and evaluating biotoxicity is the most urgent task.

The inventors have already revealed that TBT (tributyltin) and DBT (dibutyltin) in the butyltin series of substances very sensitively inhibit the P450 _SCC enzyme for converting cholesterol into pregnenolone. On the other hand, TBT has been shown to inhibit 3β-HSD, which converts pregnenolone to progesterone, and aromatase, which converts testosterone to estradiol. In addition, various heavy metals and other organic harmful substances showed a similar tendency with less sensitivity. Therefore, it was confirmed that P450 _SCC , 3β-HSD, and aromatase, which are the initial stages of the steroid production process, could be the targets of various harmful substances. In addition, some pyrethroid insecticides (deltamethrin, tetramethrin, methcyclocyclo) rather promote the activity of 3β-HSD at very low concentrations (about 0.01 μM), and phthalate compounds (Dipropyl phthalate, dibutyl phthalate) was confirmed to promote the activity of 17α-hydroxylase enzyme. Therefore, the present inventors conducted continuous research to establish a method for detecting and evaluating various harmful substances such as heavy metals, antifungal agents, insecticides, endocrine disruptors, etc. using enzymes related to steroid production and metabolism as biomarkers. . As a result, it was confirmed that the above object can be achieved simply and economically by using an amphibian, in particular, a frog follicle culture system, and the present invention has been completed.

Accordingly, a first object of the present invention is to provide a method for detecting environmentally harmful substances using a culture system of amphibian follicles.

A second object of the present invention is to provide a method for evaluating the toxicity of environmentally harmful substances using a culture system of amphibian follicles.

A third object of the present invention is to provide a method for determining the reversibility or irreversibility of environmentally harmful substance toxicity using a culture system of amphibian follicles.

A fourth object of the present invention is to provide a method for identifying steroid-producing enzymes acting on environmentally harmful substances using a culture system of amphibian follicles.

A fifth object of the present invention is to provide a kit for use in the above methods.

The first aspect of the present invention

1) Amphibian follicles are cultured in the presence of steroid production promoters and candidates;

2) quantifying the steroid generated from the follicle of step 1) to determine whether the candidate is an environmentally harmful substance:

It relates to a method for detecting environmentally harmful substances, comprising the step.

The second aspect of the present invention

1) Amphibian follicles are cultured in the presence of steroid production promoters and environmentally harmful substances;

2) To evaluate the toxicity of environmentally harmful substances by quantifying the steroid produced from the follicle of step 1):

It relates to a method for evaluating the toxicity of environmentally harmful substances, including the step.

The third aspect of the present invention

1) cultivating amphibian follicles in the presence of environmentally harmful substances, and then removing the environmentally harmful substances;

2) incubating the follicles of step 1) in the presence of a steroid production promoter;

3) Quantifying the steroid produced from the follicles of step 2) to determine whether the toxicity of the environmentally harmful substance is reversible or irreversible:

It relates to a method for determining the reversibility or irreversibility of the environmental toxicity, comprising the step.

The fourth aspect of the present invention

1) Amphibian follicles are cultured in the presence of environmentally harmful and precursor steroids;

2) Identifying the steroid-producing enzymes acting as environmentally harmful substances by quantifying the product steroid generated from the follicle of step 1):

It relates to a method for identifying a steroid-producing enzyme acts on environmentally harmful substances, including the step.

The fifth aspect of the present invention

1) amphibian follicles;

2) medium for culturing follicles; And

3) Means of Quantifying Steroids Generated from Follicle:

It relates to a kit for use in the method, comprising.

In the present invention, the amphibian is preferably a frog, and the environmentally harmful substance may be a heavy metal, an antifungal agent, an insecticide or an endocrine barrier, in particular butyltin, an imidazole-based antifungal agent, a pyrethroid-based insecticide or phthalate. In the present invention, a frog pituitary extract (Frog Pituitary Homogenate (FPH)) or a prosteroid may be used as a steroid generator.

Hereinafter, the present invention will be described in detail.

Steroid hormones produced and secreted by follicles in the ovary play a crucial role in regulating the growth, maturation and ovulation of the ovum, as well as in the reproductive cycle of animals. It also plays a fundamental role in reproductive activity by engaging in the development and maintenance of reproductive organs. Steroid hormone production of these follicles is made up of estradiol in stages by a series of enzymatic reactions using cholesterol as an initial precursor (see Figure 1). Therefore, when the activity of all steroid-producing enzymes in the follicles is precisely maintained, the reproductive activity of the animal can be normally performed.

On the other hand, the follicles extracted from amphibians, such as frog ovaries, can survive in simple cultures containing inorganic salts, and have the ability to produce large amounts of various steroids even under simple culture conditions. That is, the frog's follicle contains all of the enzymes involved in steroid production, such as 3β-HSD, 17α-hydroxylase, C _17,20 -lyase, 17β-HSD and aromatase. The present inventors have found that endocrine disruptors strongly inhibit the steroid production of follicles, and based on this, developed and established a method for detecting environmentally harmful substances and evaluating toxicity. More specifically, the present invention includes the following methods:

1) Detection of environmentally harmful substances and evaluation of toxicity that affect steroid production of follicles

2) Reversible or irreversible determination method of environmentally harmful substances

3) Identification method of steroid-producing enzyme acting environmentally harmful substances

1-a) Detection of Hazardous Candidates

The follicle consists of an egg and follicle cells surrounding it. The production of steroids is caused by the somatic cells, ie follicle cells and capillary cell layers, by stimulation of the pituitary hormone. In the present invention, amphibians, in particular frog follicles are treated simultaneously with pituitary hormone and the candidate material for a predetermined time, for example, 12 hours, artificial culture, and then secreted into the culture solution of pregnenolone, for example, radioimmunoassay ( RIA) method, by quantitative analysis, primarily detects harmful substances that inhibit steroid production of follicles (see FIG. 2).

1-b) Toxicity Evaluation of Detected Hazardous Substances

In order to evaluate the toxicity of the first detected harmful substances and similar compounds, their initial toxicity concentrations (ED ₂₀ ) and ED ₅₀ concentrations are determined. For this purpose, the follicles are artificially cultured in a culture solution containing pituitary hormones and harmful substances for different concentrations, and then quantitatively analyzed for progenolone secreted into the culture medium, for example, by RIA method. From the results obtained therefrom, for example, the Prix (Grapad) program is used to determine the initial toxicity and ED ₅₀ concentration at which the harmful substances and the compounds are affected.

2) Reversible or irreversible determination of toxic substance toxicity

It is very important to find out whether the toxicity of environmentally hazardous substances is reversible or irreversible. This is because it can be an important criterion for determining whether or not a certain period of exposure to hazardous substances can be tolerated. Therefore, the present invention provides a method for determining whether or not the reversibility of the toxic substance toxicity. To this end, the amphibian follicles are exposed to environmentally harmful substances for a certain time, the follicles are transferred to the basic culture solution, and the follicles are repeatedly washed to completely remove the harmful substances. , For example, by RIA method (see FIG. 3).

3) Detection of steroid synthase with harmful substances

The amino acid sequences of the steroid synthase present in the adrenal glands and gonads are similar in all identified species, and in particular the steroid binding sites are known to be nearly identical in sequence. This suggests that if a toxic substance acts on steroid-producing enzymes, it could affect a wide range of species without being limited to that species. In the present invention, it is revealed whether the harmful substance directly affects the activity of the steroid-producing enzyme, and if it affects, it checks which stage the steroid-producing enzyme is sensitive to. To this end, pro-steroids are added to the culture and the activity of the steroid-producing enzymes in the follicles is indirectly examined by examining whether the follicles in the culture can be converted to product steroids. The follicles are cultured for a certain period of time in a culture medium containing the harmful substances and the prosteroids of each steroid generating enzyme, and then the product steroid secreted into the culture medium is quantitatively analyzed, for example, by RIA method. Steroid synthase sensitive to the harmful substances is selected by comparing the concentrations of the harmful substances affecting the activity of each enzyme (see FIG. 4).

Hereinafter, the present invention will be described in more detail with reference to Examples, which are intended to aid the understanding of the present invention but do not limit the scope of the present invention in any way.

Example 1 Detection of Toxic Chemicals and Toxicity Evaluation Affecting Steroid Production in Follicles

Female bullfrog was collected and slaughtered by head dissection, and ovaries were removed under a dissecting microscope. The isolated ovaries are transferred to a Petri dish and washed well with Amphibian Ringer (AR) solution (6.6 g NaCl / L; 0.15 g CaCl ₂ / L; 0.15 g KCl / L), followed by two microtweezers from the ovary. Individual follicles were separated. The separated follicles were placed in each well of a porous culture dish (24 well Petri dish) each containing 1 ml of culture medium, and then the culture dish was transferred to a shaker (24 ° C., 80 rpm) and cultured for 18 hours. At this time, sodium penicillin G (30 mg / L), streptomycin (50 mg / L) and sodium bicarbonate buffer (200 mg NaHC0 ₃ / L) were added to the AR solution immediately before use. Pituitary gland extract (FPH), an AR solution (gland / ml) that extracts the pituitary gland immediately from the head of an incised frog with micro-tweezers, stores it in a freezer (-70 ° C), and collects the pituitary gland to maintain 4 ° C. After transfer to the homogenizer (glass homogenizer). The homogenized sample was centrifuged (8,000 rpm) for 20 minutes at 4 ° C. to remove solids, then dispensed in 1 ml and stored in a freezer, and dissolved as needed. When culturing follicles, FPH (0.1 gland / ml) or prosteroid (100 ng / ml) was added to promote the production of steroids in the follicles. In order to investigate the effects of environmentally harmful substances, a certain concentration (0.01 to 100 μM) of environmentally harmful substances (organic tin, heavy metals, imidazole antifungals, pyrethroid insecticides) is added to the culture medium in addition to FPH or prodrugs. The cultures were incubated for 18 hours. Unlike other steroids, pregnenolone (P5) is not secreted into the culture medium, so after incubation, remove the culture medium from the culture plate and add 1 ml of methanol per 10 follicles (1 ml per well) to remove the steroid contained in the follicles. After extraction, the inhibitory effect of P450 _SCC enzyme activity of these harmful substances was investigated by measuring the level of P5 produced using the RIA method.

The results for the organotin, heavy metal and imidazole-based antifungal agents are shown in FIG. 5. As shown in FIG. 5 (A), tributyltin (TBT) and dibutyltin (DBT) of butyltin compounds most effectively inhibited P5 production of follicles by stimulation of pituitary hormone, and especially TBT was 0.05 μM ( ED ₅₀ ) significantly inhibited P5 production of follicles. In the case of phenyltin compounds, mono- (MPT), di- (DPT), and triphenyltin (TPT) all inhibited the formation of P5 in follicles, while ED ₅₀ of DPT and TPT was 0.05 μM, and ED ₅₀ of MPT was 0.18 μM. (FIG. 5 (B)). These results show that the frog's P450 _SCC enzyme system is extremely sensitive to TBT and DBT, which is a good indicator for the determination of harmful substances in this family.

In addition, ketoconazole, itraconazole and cloconazole of the imidazole family of compounds significantly inhibited the P5 production of follicles by stimulation of the pituitary hormone, but fluconazole, myconazole and echonazol had no effect. In particular, the ED ₅₀ of ketoconazole, itraconazole and cloconazole, which inhibit the production of follicles P5, exhibited 0.06 μM, 0.7 μM and 5 μM, respectively, so among the antifungal agents used in this example, cloconazole was most effective in the follicle P450 _SCC. It was shown to inhibit enzymatic activity (Figs. 5 (C) and (D)). The results show that the method can be usefully used for the toxicity evaluation of imidazole compounds.

On the other hand, as a result of investigating the effects of various heavy metals, zinc and lead have the effect of inhibiting the generation of P5 of the follicles (Fig. 5 (E)), cadmium has the effect of promoting P5 production at low concentrations, at high concentrations The dual effect of inhibiting P5 production was shown (FIG. 5 (G)). However, cobalt and arsenic did not affect the steroid production of follicles (Fig. 5 (F)). Therefore, the P450 _SCC enzyme system was judged to be somewhat inefficient in determining the effect of heavy metals. However, the fact that cadmium promotes the production of P5 at very low concentrations is a very interesting result, suggesting the possibility of using this part as a biomarker.

The results for the pyrethroid family of insecticides are shown in Table 1.

Effects of Pesticides on Steroid Production in Follicles 0.01 μM 0.1 μM 1 μM 10 μM 100 μM BioAlltrin + ^{* 1} + + + + Dimethoate + + + + + Pentachlorophenol + + + ++ + Permethrin + ++ ++ +++ + Tetramethrin + ++ ++ ++ + Deltamethrin ++ + ++ ++ ++ Methcyclocyclo ++ + + + ++ Penvalerate + + - ++ +

*One; Maintain almost similar level of activity compared to the control

+; 110 ~ 200% higher enzyme activity than control

++; 201 ~ 300% higher enzyme activity than control

As shown in Table 1, the pyrethroid-type insecticides rather showed an effect of slightly increasing the steroid (P5) production of follicles by stimulation of pituitary hormone. These results indicate that pyrethroid-based insecticides promote the activity of the P450 _SCC enzyme, suggesting the possibility of using it as a biomarker.

Taken together, we can see that:

1) Inhibition of P450 _SCC enzyme activity in frog follicles can be used as a biomarker for the determination of butyltin-based harmful substances. TBT and DBT are more toxic than MPT.

2) Investigation of six imidazole antifungal agents showed that ketoconazole, itraconazole and cloconazole inhibit the activity of P450 _SCC enzyme more effectively than others.

Investigation of five heavy metals revealed that zinc and lead inhibit P450 _SCC enzyme activity more effectively than other heavy metals.

4) Investigations of eight pyrethroid-based insecticides showed that some insecticides rather promote the activity of the P450 _SCC enzyme.

These results show that the use of follicle steroids in the reproductive system of amphibians facilitates the detection and toxicity assessment of environmentally harmful substances that affect sex hormone production.

Example 2: Reversibility and irreversibility determination of toxicity of environmentally harmful substances

To determine whether the toxicity of the environmentally hazardous substance is reversible or irreversible, the bullfrog follicle is exposed to the environmentally hazardous substance (100 μM) for a period of time (5, 30, 60, 120, 240, 360 minutes), and then the follicle is exposed. After removing the harmful substances completely by transferring to the basal culture, and repeatedly washed, the pituitary hormone-treated medium was incubated for 18 hours, and the P5 level generated from the follicles was measured using the RIA method.

The results are shown in FIG. Among the butyltin compounds, DBT, TBT, and tetrabutyltin (TTBT) were very toxic, and it was confirmed that the follicle steroid production was completely inhibited even when the follicles were exposed to these substances for 30 minutes (Fig. 6 (A)). In addition, even when exposed to a phenyltin compound for a very short time (5 minutes) it was confirmed that the steroid production of the follicles is suppressed, and the irreversible toxicity is strong in the order of TPT> DPT> MPT (Fig. 6 (B)). However, MBT did not inhibit the steroid production of follicles even after 4 hours of exposure (FIG. 6 (A)). These results indicate that this model can be used as a very useful biomarker for determining the reversibility of the toxic effects of butyltin and phenyltin.

Ketoconazole, itraconazole and econazol among the various imidazole series compounds were confirmed to inhibit the steroid production of the follicles even after about 30 minutes of exposure (FIG. 6 (C)). In addition, it was confirmed that cadmium, zinc, and lead among various kinds of heavy metal compounds are relatively irreversibly toxic compared to mercury and cobalt. It could be confirmed (FIG. 6 (E) and (F)). These results show that environmentally harmful substances have an irreversible toxic effect even after relatively short exposures. These results show that the method can be fully utilized to determine the reversibility of toxicity when living organisms are exposed to environmentally harmful substances.

Example 3: Identification of steroid synthase acting on environmentally harmful substances

In this example, it was intended to investigate whether the identified harmful substances deterministically inhibit or promote the action of the steroid-producing enzyme of the follicle. To this end, several environmentally harmful substances (100 μM), which have already been identified as harmful, were first treated in the follicles and incubated for 18 hours, and then the produced and secreted steroids were quantitatively analyzed by the RIA method. Was investigated. All steroids other than P5 were well secreted into the cultures, so the steroid concentrations in the cultures could be measured directly without additional extraction.

The results are shown in FIGS. 7 to 9. Among the various butyltin compounds, in particular TBT inhibits the activity of 3β-HSD, 17β-HSD and aromatase (Figs. 7 (A), (D) and (E)) and _inhibits the activity of C _17,20 -lyase. Partially inhibited (FIG. 7C), DBT and TTBT inhibited the activity of these steroid synthase somewhat less strongly than TBT, but did not inhibit the activity of 17α-hydroxylase unlike TBT (FIG. 7 (C). B)). In contrast, MBT did not affect the activity of almost all steroid synthase (FIGS. 7 (A) to (E)). These results suggest that the target of the steroid-inhibiting effect of TBT is not on any specific enzyme, but on several enzymes, but also does not affect some enzymatic actions.

Among the imidazole compounds, ketoconazole has been shown to promote the activity of C17,20-lyase, which converts 17α-OH-progesterone to androstenedione, and fluconazole and econazole also enhance the activity of this enzyme. Some acceleration could be observed (FIGS. 8A and 8B). It was also confirmed that the imidazole family of compounds used in this study did not affect the activity of 17β-HSD, which converts androstenedione (AD) to testosterone (T) (Figs. 8 (C) and ( D)). These results show that imidazole compounds have a facilitating effect rather than inhibiting the action of some enzymes in steroid synthesis.

First, we investigated whether heavy metals affect the activity of HSD in the cytoplasm. Mercury and cadmium, among other heavy metals, selectively inhibited the activity of 17β-HSD, an enzyme that converts androstenedione to testosterone, and lead partially inhibited the activity of the enzyme (Figs. 9 (A) and (B)). ). However, heavy metals such as cadmium had a very small effect on the activity of 3β-HSD, which converts pregnenolone (P5) to progesterone (P4) (Figs. 9 (C) and (D)).

The results for the phthalate compounds are shown in Table 2.

o; Maintain almost similar level of activity compared to the control

+; 110 ~ 200% higher enzyme activity than control

++; 201 ~ 300% higher enzyme activity than control

As shown in Table 2, the phthalate-based compound had a somewhat synergistic effect rather than inhibiting the activity of the steroid synthase of follicles. In particular, dipropylphthalate (DPrP) and di-n-hexylphthalate (DHP) showed the effect of enhancing the activity of 17α-hydroxylase, which converts progesterone to 17α-OH-progesterone, but other phthalate compounds There was a slight increase in the activity of steroid synthase in follicles.

Using the steroid-producing system of amphibian follicles from the above results, it is possible not only to determine which environmentally harmful substances affect the activity of steroid-producing enzymes, and to predict the damage accordingly.

In the present invention, an amphibian capable of producing various steroid hormones, particularly a follicle culture system of frogs, has been developed and established a method for detecting environmentally harmful substances affecting sex hormone production and evaluating its toxicity. The method according to the present invention is simple and economical by using a culture system of amphibian follicles, which is simple to culture and excellent in steroid production ability, and can objectively and scientifically detect and evaluate toxicity of environmentally harmful substances affecting the reproduction of animals. It can be used in many ways, and can provide basic data for judging the impact on human health.

Claims (8)

1) Amphibian follicles are cultured in the presence of steroid production promoters and candidates; 2) quantifying the steroid generated from the follicle of step 1) to determine whether the candidate is an environmentally harmful substance: A method for detecting environmentally harmful substances, comprising the step of: 1) Amphibian follicles are cultured in the presence of steroid production promoters and environmentally harmful substances; 2) To evaluate the toxicity of environmentally harmful substances by quantifying the steroid produced from the follicle of step 1): A method for assessing the toxicity of environmentally harmful substances, comprising the steps of: 1) cultivating amphibian follicles in the presence of environmentally harmful substances, and then removing the environmentally harmful substances; 2) incubating the follicles of step 1) in the presence of a steroid production promoter; 3) Quantifying the steroid produced from the follicles of step 2) to determine whether the toxicity of the environmentally harmful substance is reversible or irreversible: Reversible or irreversible determination method of the environmental toxicity, comprising the step. 1) Amphibian follicles are cultured in the presence of environmentally harmful substances and prosteroids; 2) Identifying the steroid-producing enzymes acting as environmentally harmful substances by quantifying the product steroid generated from the follicle of step 1): A method for identifying a steroid-producing enzyme acting on environmentally harmful substances, comprising the step. The method according to any one of claims 1 to 4, wherein the amphibian is a frog. The method according to any one of claims 1 to 4, wherein the environmentally harmful substance is a heavy metal, an antifungal agent, an insecticide or an endocrine disruptor. 7. The method according to claim 6, wherein the environmentally harmful substance is butyltin, imidazole-based antifungal agent, pyrethroid-based insecticide or phthalate. 1) amphibian follicles; 2) medium for culturing follicles; And 3) Means of Quantifying Steroids Generated from Follicle: Kits for use in the method according to any one of claims 1 to 4, comprising.

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