KR102160793B1 - Composition for relieving reproductive toxicity by bisphenol A comprising antioxidant material - Google Patents

Abstract

The present invention relates to a composition for alleviating reproductive toxicity by bisphenol A (BPA) containing an antioxidant as an active ingredient, and the antioxidant glutathione (GSH; gamma-glutamylcysteinylglycine), vitamin C according to the present invention (vitamin C, Vit C; L-ascorbic acid), or vitamin E (vitamin E, Vit E; α-tocopherol) is produced by bisphenol A, the production of reactive oxygen species, protein kinase A (protein kinase). A; PKA) by showing the effect of alleviating reproductive toxicity such as activation, protein tyrosine nitration, reduction of sperm fertility, and early embryonic development ability of sperm, making it useful as a composition for alleviating reproductive toxicity by bisphenol A. It is expected to be available.

Description

Composition for relieving reproductive toxicity by bisphenol A comprising antioxidant material}

The present invention is a composition for alleviating reproductive toxicity by bisphenol A containing an antioxidant as an active ingredient, specifically, at least one selected from the group consisting of glutathione, vitamin C, and vitamin E. It relates to a composition for reproductive toxicity alleviation by bisphenol A (Bisphenol-A; BPA) comprising an antioxidant as an active ingredient.

Bisphenol A (BPA; 2,2-bis(4-hydroxyphenyl) propane) is an endocrine disrupting substance widely used in the plastics industry as a representative environmental hormone, and it is an endogenous hormone by mimicking or antagonizing hormone activity in the body. Synthesis, metabolism, transport and elimination processes. These environmental hormones, unlike biological hormones, are not easily degraded, remain in the environment and body for several years, and are reported to have a property of being concentrated in fats and tissues of living organisms such as the human body. In a recent study, BPA has been reported to cause developmental, systemic, neurological, immune and reproductive disorders, and as a reproductive toxic substance, it is reported that steroids are produced, embryonic developmental defects, testes/epidermal function loss, abnormal accessory glands function, etc., resulting in decreased fertility and infertility Cause of. The easiest way to avoid BPA toxicity is to minimize exposure, but humans can be exposed to BPA anywhere through the oral, respiratory and dermal routes, and limiting their use in everyday life is practically difficult. Therefore, there is a need to develop potential therapeutic strategies to minimize BPA toxicity.

BPA acts as a selective estrogen receptor modulator by binding to estrogen receptors as exogenous estrogens and causing a similar effect, binding to genomic (nuclear and cytoplasmic) and non-genomic (membrane-bound) estrogen receptors (ER) in cells. . In addition, BPA acts as an activator of estrogen-related receptor gamma and growth factor receptors, antagonizes thyroid hormone receptors, and has anti-androgen properties. Since mature sperm contains most of these receptors (e.g. ERα, ERβ, growth factor and androgen receptors), sperm potentially test the effects/molecular mechanisms of BPA action as well as subsequent effects on male fertility/reproduction. Suitable for

Recently, exposure of mouse sperm to 100 μM of BPA showed rapid estrogen signal (vigenomic) by activation of several kinase systems, and changes such as decreased motility, acrosome response/fertilization in sperm exposed to BPA, Changes in mitochondrial activity, fertilization and reduction in embryonic development were observed. In addition, a change similar to the above was measured in F1 sperm after exposure to BPA during pregnancy. These detrimental effects were mainly caused by increased oxidative stress caused by increased reactive oxygen species (ROS) in sperm exposed to BPA. Oxidative stress increased by BPA was also measured in other studies on neurons and kidney cells. Low levels of ROS are important for the regulation of normal sperm function, such as acquisition of fertility, activation of acrosome response and motility, and excessive oxidative stress negatively affects the lipid, protein and DNA levels of sperm, resulting in infertility.

On the other hand, antioxidants are molecules that inhibit oxidation to prevent oxidative stress or overproduction of ROS in cells and tissues.In conventional studies, ingestion of dietary antioxidants such as selenium, vitamin E, and Lepidium meyenii inhibits the production of ROS in sperm. It was found to have a protective effect on the sperm quality of stallions and dogs. In addition, glutathione peroxidase, superoxide dismutase, cysteine, apigenin and ferulic acid have been reported to reduce the oxidative stress of sperm during cryopreservation. And had a positive effect on sperm motility and fertilization.

Accordingly, the present inventors tried to determine how the above-described antioxidants affect the stress caused by BPA in sperm exposed to BPA.

Korean Patent Publication No. 10-2018-0102829

In vitro , antioxidants such as glutathione (GSH; gamma-glutamylcysteinylglycine), vitamin C (vitamin C, Vit C; L-ascorbic acid), and vitamin E (vitamin E, Vit E; α-tocopherol) are It was confirmed that it relieves the stress of sperm exposed to BPA, and the present invention was completed based on this.

Accordingly, the problem to be solved by the present invention is to provide a composition for alleviating reproductive toxicity by bisphenol-A (BPA), comprising an antioxidant as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned can be clearly understood by those of ordinary skill in the technical field to which the present invention belongs from the following description. There will be.

In order to achieve the above object, the present invention provides a composition for alleviating reproductive toxicity by bisphenol A (Bisphenol-A; BPA), comprising an antioxidant as an active ingredient.

In addition, the present invention provides a food composition for preventing or improving reproductive toxicity by bisphenol-A (BPA), comprising an antioxidant as an active ingredient.

As an embodiment of the present invention, the antioxidant may be selected from the group consisting of glutathione, vitamin C, and vitamin E.

In another embodiment of the present invention, the concentration of glutathione may be 1mM to 10mM.

As another embodiment of the present invention, the concentration of vitamin C (vitamin C) may be 25 μM to 250 μM.

As another embodiment of the present invention, the concentration of vitamin E may be 1mM to 10mM.

In another embodiment of the present invention, the reproductive toxicity caused by the bisphenol A is reactive oxygen species generation, protein kinase A (PKA) activation, protein tyrosine nitration, reduction in sperm fertility acquisition, or It may be any one or more of the decline in early embryonic development ability.

In addition, the present invention provides a method for alleviating reproductive toxicity by bisphenol A (BPA), comprising administering the composition to an individual.

In addition, the present invention provides the use of the composition to relieve reproductive toxicity by Bisphenol-A (BPA).

Antioxidant substances according to the present invention glutathione (glutathione, GSH; gamma-glutamylcysteinylglycine), vitamin C (vitamin C, Vit C; L-ascorbic acid), or vitamin E (vitamin E, Vit E; α-tocopherol) is bisphenol A ( Reproductive toxicity, such as generation of reactive oxygen species caused by bisphenol A), activation of protein kinase A (PKA), nitrosation of protein tyrosine, reduction of sperm fertility, and early embryonic development of sperm It is expected that it can be usefully used as a composition for alleviating reproductive toxicity by bisphenol A by exhibiting such an effect.

1A is a view showing the structure of bisphenol A (BPA) according to an embodiment of the present invention.
1B is a view showing the structure of glutathione (GSH) according to an embodiment of the present invention.
1C is a view showing the structure of vitamin C (Vit C) according to an embodiment of the present invention.
1D is a view showing the structure of vitamin E (Vit E) according to an embodiment of the present invention.
1E is a diagram showing a three-dimensional structure of bisphenol A (BPA) according to an embodiment of the present invention.
2 is a view showing the effect of each concentration of antioxidants (GSH, Vit C, Vit E) on sperm motility according to an embodiment of the present invention.
3A to 3C are diagrams showing the effect of antioxidants on the aspect of obtaining fertility in BPA-exposed sperm according to an embodiment of the present invention.
4A to 4D are diagrams showing the effect of antioxidants on protein kinase-A (PKA) activity and tyrosine phosphorylation in BPA-exposed sperm according to an embodiment of the present invention.
5A to 5C are diagrams showing the effect of antioxidants on intracellular ROS and protein tyrosine nitration in BPA-exposed sperm according to an embodiment of the present invention.
6 is a view showing the effect of an antioxidant on fertilization and early embryo development ability in BPA-exposed sperm according to an embodiment of the present invention.

The present invention provides a composition for reproductive toxicity relief by bisphenol A (Bisphenol-A; BPA), comprising an antioxidant as an active ingredient.

The term "Bisphenol-A, BPA; 2,2-bis(4-hydroxyphenyl) propane)" used in the present invention is an aromatic compound composed of two phenols having an alcohol group on a benzene ring, such as polycarbonate or epoxy resin. It is used as a raw material for plastic production and is an environmental compound with estrogen activity, xenoestrogen, which reduces male genital development and semen quality.

In the present invention, it was confirmed that 100 μM of the bisphenol A adversely affects various sperm functions and fertilization ability, and 100 μM of BPA was used in the experiment. Exposure of mouse sperm to the 100 μM BPA can change mitochondrial activity and reduce cellular ATP levels. Physiologically, mitochondria regulate key actions such as the production, detoxification and cell death of ATP and reactive oxygen species, and mitochondrial reactive oxygen species are maintained at healthy concentrations to maintain the balance of intracellular antioxidant defense and delicate interactions. Should be. Therefore, antioxidants can improve mitochondrial function and deliver optimal ATP for sperm motility in a BPA-treated microenvironment.

The term "antioxidant" used in the present invention serves to prevent oxidative stress in cells and tissues as a substance that prevents excessive production of active oxygen. These antioxidants neutralize free radicals that cause harmful reactions in cells before attacking DNA or oxidizing lipids. In addition, antioxidants are highly reactive and react with harmful substances in the body to prevent chain reactions caused by free radicals, thereby protecting cells.

In the present invention, the antioxidant may be selected from the group consisting of glutathione, vitamin C, and vitamin E, but is not limited thereto.

The term "glutathione (GSH; gamma-glutamylcysteinylglycine)" used in the present invention is a kind of peptide and is a structure of γ-L-glutamyl-L-cysteinylglycine, and the glutamyl group is a γ-peptide structure. It is different from peptides. Glutathione is not only related to cellular functions such as intracellular redox activity, xenobiotic metabolism, intracellular signaling, and gene regulation, but also cells that can be induced by free radicals and free radicals. It is especially important as an amino acid that protects against damage. More specifically, glutathione is reversibly changed to glutathione disulfide (GSSG), which is an oxidized form of glutathione in which glutathione of two molecules is linked through disulfide (-SS-) bonds by reacting with oxidants in vivo. To regulate the oxidation-reduction potential in vivo.

The term "vitamin C" used in the present invention has a chemical formula of C ₆ H ₈ O ₆ and is one of the trace elements for maintaining the function and health of the human body, and is also referred to as ascorbic acid (L-ascorbic acid). It helps the human body to resist infection, heal wounds and keep tissues healthy, and as one of antioxidants, it prevents cell damage by free radicals.

The term "vitamin E" used in the present invention has a formula of C ₂₉ H ₅₀ O ₂ and is also referred to as tocopherol or tocotrienol, and the tocopherol and tocotrienol are each α, β, γ, It can have four forms such as δ tocopherol, α, β, γ, and δ tocotrienol. The vitamin E directly reduces free radicals generated through ascorbate-induced lipid peroxidation, particularly peroxyl groups and alkoxyl groups (ROO·), thereby reducing intracellular oxidative stress and restoring the integrity of the acrosome membrane. In addition, it is involved in the maintenance of reproductive function and muscle function, and if it is insufficient, reproductive function decline, anesthesia, abortion, infertility, anemia, and muscle atrophy occur. Compared to other fat-soluble vitamins, the toxicity is relatively low, and thus hyperactivity does not appear well.

In the present invention, the concentrations of glutathione, vitamin C, and vitamin E may be 1mM to 10mM, 25μM to 250μM, and 1mM to 10mM, respectively, preferably each concentration is When 5mM, 100μM, 2mM can have the most positive effect on sperm motility, it can show the greatest alleviation effect on reproductive toxicity by bisphenol A, but is not limited to the concentration.

In the present invention, the reproductive toxicity caused by the bisphenol A is reactive oxygen species generation, protein kinase A (PKA) activation, protein tyrosine nitration, decreased sperm fertility acquisition, or early embryonic development ability of sperm It may be one or more of the reductions, but is not limited thereto.

The term "reactive oxygen species (ROS)" as used in the present invention refers to a chemically reactive molecule containing an oxygen atom. The molecules contain oxygen ions and hydrogen peroxide, and because of unpaired electrons It is very reactive. Reactive oxygen species play a variety of roles in our body, examples of which include apoptosis, genetic expression, and intracellular signaling cascades. However, if these reactive oxygen species are present in the body at a concentration higher than necessary, fat, protein, and DNA in the body will be damaged. When cells are deformed or killed through such damage, it can cause various diseases, such as cancer, brain degenerative diseases, and heart disease.

The term "capacitation" as used in the present invention refers to specific changes such as maturation or activity and processes that sperm undergo in order to develop the ability to fertilize in an egg. The ability to modify is significantly reduced. Acquisition of fertility can be confirmed through changes occurring inside sperm, for example, changes in mitochondria tissue in the tail of sperm or changes occurring on the cell surface. Corrective power acquisition includes physical characteristics (total motor capacity, forward motor capacity, rapid motor capacity), kinematic parameters (linear speed, average path speed, curved speed and average displacement of the lateral head) and channel activator, A23187 ionophore ( It can be measured by conversion into an acrosome reaction ability caused by stimulation by ionophore, etc.

The term "acrosome reaction" used in the present invention refers to a reaction that occurs in the acrosome while the sperm approaches the zona pellucida of the egg, and this reaction surrounds the acrosome of the sperm when the sperm approaches the egg. The membrane is fused with the plasma membrane of sperm to secrete contents containing enzymes inside the acrosome, thereby allowing the sperm to fuse with the egg.

In the present invention, the composition comprising the antioxidant as an active ingredient may be provided as a pharmaceutical composition for preventing or treating reproductive toxicity caused by bisphenol A.

The term “prevention” as used in the present invention refers to any action that suppresses reproductive toxicity or delays onset by administration of the pharmaceutical composition according to the present invention.

The term “treatment” used in the present invention refers to any action in which symptoms of reproductive toxicity are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

In the present invention, the "pharmaceutical composition" means that it is prepared for the purpose of preventing or treating diseases, and each may be formulated and used in various forms according to conventional methods. For example, it can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc., creams, gels, patches, sprays, ointments, warnings, lotions, liniment, pasta or It can be formulated and used in the form of external preparations for skin such as cataplasma, suppositories, and sterile injectable solutions.

The pharmaceutical composition according to the present invention may further include suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. In this case, as carriers, excipients, and diluents that may be included in the composition, lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium Silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient for the antioxidant substance such as starch, calcium carbonate, sucrose ( sucrose) or lactose (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.

The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intravenous, subcutaneous, intraperitoneal or topical application) according to a desired method, and the dosage is It depends on the degree, drug form, administration route and time, but may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, drug activity of the patient, Sensitivity to drugs, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field can be determined. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art. Administration may be administered once a day, or may be divided several times.

In the present invention, the composition comprising the antioxidant as an active ingredient may also be provided as a food composition for preventing or improving reproductive toxicity caused by bisphenol A.

In the present invention, the food composition may be a health functional food composition, but is not limited thereto.

The term “improvement” as used herein refers to any action that at least reduces the severity of a parameter related to the condition being treated, for example, symptoms.

The term "health functional food composition" used in the present invention is formulated as one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations including at least one of carriers, diluents, excipients and additives. It is characterized by being.

Foods that can be added to the antioxidants of the present invention include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods. Additives that may be further included in the present invention include natural carbohydrates, flavoring agents, nutrients, vitamins, minerals (electrolytes), flavoring agents (synthetic flavoring agents, natural flavoring agents, etc.), coloring agents, fillers, lactic acids and salts thereof, Alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, antioxidants, glycerin, alcohols, carbonation agents, and at least one component selected from the group consisting of pulp may be used. Examples of the above-described natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

In addition to the above, the composition according to the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickeners, lactic acid and salts thereof, alginic acid and salts thereof, organic acids, protection It may contain colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like.

In addition, the composition according to the present invention may contain pulp for the manufacture of natural fruit juices and vegetable beverages. These components may be used independently or in combination.

Specific examples of the carrier, excipient, diluent, and additive are, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, The group consisting of microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, and mineral oil It is preferable to use at least one selected from.

In addition, the present invention provides a method for alleviating reproductive toxicity by bisphenol A (BPA), comprising administering the composition to an individual.

In addition, the present invention provides the use of the composition to relieve reproductive toxicity by Bisphenol-A (BPA).

The term "individual" as used in the present invention means a subject in need of treatment of a disease, and more specifically, human or non-human primates, mice, dogs, cats, horses, and cattle. Means mammal.

As used herein, the term "administering" means providing a given composition of the present invention to an individual in any suitable manner.

In one experimental example of the present invention, as a result of confirming the effect of antioxidants on the motility parameters of sperm exposed to BPA, it was confirmed that sperm exposed to 100 μM of BPA decreased motility compared to the control group, and treated with GSH and Vit E. When the motility was recovered, it was confirmed that the Vit C group did not (see Experimental Example 1).

In another experimental example of the present invention, as a result of confirming the effect of the antioxidant on the aspect of obtaining fertility of sperm exposed to BPA, the number of acrosome-reactive (AR) cells in BPA-exposed sperm cultured with GSH or Vit E was compared with the control level. A similar thing was confirmed (see Experimental Example 2).

Mammalian sperm have to undergo a series of physiological and biochemical modifications in the female reproductive organs for fertilization, which can also be done in vitro in special media containing appropriate concentrations of electrolytes, energy and proteins. The sperm that has acquired fertility causes excessive activation, causes an acrosome reaction, penetrates the zona pellucida, and fuses with oocytes. At the molecular level, fertilization and acrosome reactions are associated with increased sperm membrane fluidity, regulation of intracellular ionic equilibrium, and activation of soluble adenylyl cyclase, cAMP and PKA. In addition, activated PKA phosphorylates several target proteins (related to fertilization acquisition) primarily at tyrosine residues in sperm.

Accordingly, in another experimental example of the present invention, as a result of confirming the effect of the antioxidant on protein kinase-A (PKA) activity and tyrosine phosphorylation of BPA-exposed sperm, PKA activity increased at 16 kDa and increased at 60 kDa by BPA exposure. It was confirmed that tyrosine phosphorylation was reduced by treatment with GSH, Vit C, and Vit E (see Experimental Example 3).

In another experimental example of the present invention, as a result of confirming the effect of antioxidants on ROS and protein tyrosine nitration in the cells of sperm exposed to BPA, ROS increased by BPA exposure was affected by the treatment of GSH, Vit C, and Vit E. It was confirmed that tyrosine nitration, which was reduced by and increased by BPA exposure, was reduced by treatment with GSH and Vit C (see Experimental Example 4).

In another experimental example of the present invention, as a result of confirming the effect of antioxidants on the fertilization and early embryo development ability of sperm exposed to BPA, GSH and Vit E can restore the fertilization and early embryo development ability of sperm exposed to BPA. It was confirmed that it can be (see Experimental Example 5).

Hereinafter, preferred examples and experimental examples are presented to aid in understanding the present invention. However, the following Examples and Experimental Examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following Examples and Experimental Examples.

Example 1. Material preparation

1-1. Experimental animals Preparations

Ethical approval for animal studies of the present invention was made by the Institutional Animal Care and Use Committee (IACUC Number: 2016-00009) of Chung-Ang University.

For the experiment, specimens of 8-week-old CD-1 (ICR) male mice purchased from Daehan BioLink® (Eumseong, Chungcheongbuk-do) were collected and acclimated for at least 1 week before subsequent experiments, at which time the mice were allowed to stay for 12 hours by day. /It was acclimated in a room for 12 hours at night, temperature 20-25°C, humidity 50-60%. In addition, commercial mouse pills and glass bottles of water were freely provided during the experiment, and maximum precautions were taken in the animal's room facility to minimize external BPA exposure.

1-2. Reagent and culture solution

All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sperm basic media (BM) is modified Tyrode medium (97.84 mM NaCl, 1.42 mM KCl, 0.47 mM MgCl ₂ .H ₂ O, 0.36 mM NaH ₂ PO ₄ H ₂ O, 5.56 mM D-glucose, 25 mM NaHCO ₃ , 1.78 mM CaCl ₂ H ₂ O, 24.9 mM Na-lactate, and 50 μg/ml gentamycin) were used. The BM was supplemented with 0.4% bovine serum albumin (BSA) to induce fertility acquisition, and pre-incubated at 37°C in the presence of 5% CO ₂ in air (Rahman et al., 2015, 2017). In addition, the pH and osmotic pressure of BM were maintained at 7.2 ± 0.2 and 300 ± 20 mOsm/kg, respectively (Ryu et al., 2017b), and both BPA and antioxidants were dissolved in dimethylsulfoxide (DMSO) and 1 day before the experiment. It was added to the desired molecular concentration.

1-3. Selection of bisphenol A and antioxidants and exposure to sperm

In the previous study results, it was confirmed that 100 μM of bisphenol A (BPA, see Fig. 1a) adversely affects various sperm functions and fertilization ability, and 100 μM of BPA was used in the experiment (Perlman, 2016; Rahman et al., 2015, 2016). In addition, the significant effect of this dose on the functionality of testicular germ cells (eg, germ cell viability and proliferation) and spermatogonial stem cells under in vitro culture conditions was confirmed (Karmakar et al., 2017). Because sperm contain specific transporters for BPA (Fig. 1e) and antioxidants (Williams et al., 2014; Yadav et al., 2018; Juma et al., 2017) glutathione (GSH, see Fig. 1b). ), vitamin C (vitamin C; Vit C, see Fig. 1c), and vitamin E (vitamin E; Vit E, see Fig. 1d), and three commonly used non-enzymatic antioxidants were used in the present invention.

To determine the dosage of the antioxidant, we first investigated several in silico studies on the effect of antioxidants on sperm parameters in different animal species (Donnelly et al., 2000; Shah et al., 2017; Dalvit et al. ., 2005; de Vasconcelos Franco et al., 2016). Then, using mouse sperm, it was confirmed whether these concentrations can have a positive effect on motility by considering the tentative dose for each antioxidant for preliminary experiments.

As a result, as shown in Figure 2, finally, when the concentrations of GSH, Vit C and Vit E were 5 mM, 100 μM, and 2 mM, it was confirmed that the most positive effect on sperm motility was observed. Proceeded.

1-4. Sperm collection, preparation, and antioxidants To BPA exposure

Sperm were collected on the cauda epididymis of sexually mature male mice (10-12 weeks old) according to the protocol described in the previous study (Rahman et al., 2015, 2016).

Specifically, after the mice were sacrificed, the epididymis tail was collected from each mouse, placed on a clean dry filter paper, and the surrounding fat was removed. Then, the epididymis tail was placed in a culture disk containing BM, and was pierced using a sterile needle to disperse sperm. Thereafter, preintubation was performed for 12 minutes, and the sperm suspension was added to BM supplemented with BPA alone or to BM supplemented with antioxidants different from BPA. That is, a total of five treatment groups, including the control, BPA, BPA+GSH, BPA+Vit C, and BPA+Vit E, treated at the concentration determined in Example 1-3 were used in the present invention, and the control sperm were DMSO. Treated with only. In addition, when mice sperm were treated with BPA (100 μM) and cultured, the minimum time to adversely affect sperm parameters (eg, motility and viability) was 6 hours (Rahman et al., 2015, 2016), and sperm suspension was used. Incubated for 6 hours in BM under various exposure schemes.

Example 2. Experiment method

2-1. Automatic sperm analysis (computer-assisted sperm analysis; CASA)

Sperm motility and motor dynamics according to different treatments were analyzed using the SAIS-PLUS version 10.1 CASA software program (Medical Supply, Seoul, Korea), and in previous studies (Rahman et al., 2015; Ryu et al., 2017). ), CASA was used. In each treatment group, 10 μl of sperm samples were placed in a Makler chamber (Makler, Haifa, Israel) preheated to 37° C. and a phase difference 10× objective was used to analyze sperm in the CASA program. The settings of the above program were attached when the frame was acquired as follows: Frame rate: 30 Hz, minimum contrast: 7, minimum size: 5, low/high size gate: 0.4-1.5, low/high intensity gate: 0.4 -1.5, non-motile head size: 16, non-motile brightness: 14. Using the above program, 5 representative fields with at least 250 sperm cells were randomly selected to analyze the ratio of motile sperm to kinetic dynamics.

2-2. Hoechst 33258/ chlorotetracycline (H33258/ CTC ) dyeing

The effects of BPA and antioxidants on sperm fertility acquisition were analyzed by the previously reported double staining method (Rahman et al., 2015; 2017). After incubation, the sperm sample was centrifuged at 100 × g for 2.5 minutes to remove the supernatant, and 15 µl of H33258 solution was added to 135 µl of sperm sample and kept at room temperature for 2 minutes. Then, 2% polyvinylpyrrolidone was added to the phosphate buffer solution (PBS), stained, centrifuged at 100 × g for 2.5 minutes, and the sperm pellet was resuspended in 100 µl of CTC solution and 100 µl of PBS. After staining as described above, 10 μl of sperm sample was dropped onto a glass slide with a cover glass. The aspect of fertility acquisition of sperm samples by each treatment was determined under a Microphot-FXA microscope (Epifluorescence illumination) (ultraviolet, BP 450±490/LP 515 excitation/emission filter BP 340±380/LP 425). Nikon, Tokyo, Japan). Finally, sperm with the ability to acquire fertility (B, the acrosome area and dark posterior acrosome are stained with green fluorescence), sperm with the acrosome reaction (AR, the head is not stained), or the ability to obtain fertility It was classified as missing sperm (F, sperm hair was uniformly dispersed and dyed with bright green fluorescence). At least 400 sperm were analyzed on each glass slide and each procedure was repeated at least 3 times in each treatment group.

2-3. Reactive oxygen species (reactive oxygen species; ROS ) Measure

Cellular ROS levels were assessed using the 2'-7'-dichlorofluorescein diacetate (DCFDA) kit (ab113851, Abcam, Cambridge, England) as reported in a previous study (Rahman et al., 2015, 2017). Specifically, after culturing sperm of five different treatment groups by the method of Example 1-4, the sample was washed and resuspended in a DCFDA mixture, followed by incubation at 37° C. for 30 minutes. After incubation, the sperm sample was washed with 1 ml of 1 x buffer solution. Then, the sample was washed again and resuspended in 500 μL of 1× additional buffer solution. Finally, the sperm suspension was placed in a 96-well plate, and the fluorescence of the 96-well plate was detected using the SoftMax Pro 5 software program (Molecular Devices, San Jose, CA, USA) at two excitation wavelengths of 485 nm and 535 nm. .

2-4. Western Blotting

After washing the sperm cultured under different treatment conditions by the method of Example 1-4 for 10 minutes with PBS, the supernatant was removed and the sperm pellet was reproduced in Laemmli sample buffer using 5% 2-mercaptoethanol. It was turbid (Ryu et al., 2017b; Rahman et al., 2015, 2017). The sample buffer containing 500×10 ⁶ sperm cells/mL from each treatment was electrophoresed on 12% SDS-PAGE, and then transferred to a polyvinylidene fluoride membrane (Amersham, Piscataway, NJ, USA). The membrane was then blocked with a blocker (3% ECL prime blocker, Amersham) for 3 hours, and the membrane was washed twice for 2 minutes with PBS-T. Thereafter, the membrane was incubated at 4° C. overnight with the primary antibody. The primary antibodies include anti-phospho-PKA substrate (1:2000, Cell Signaling, Danvers, MA, USA), anti-phosphotyrosines (1:2000, ab16389, Abcam), and anti-nitrotyrosines (1:1000, ab42789). , Abcam) was used. For internal regulation, α-tubulin was detected using anti-α-tubulin (1:10000, ab7291, Abcam). After incubation with the primary antibody overnight, the membrane was washed and treated with horseradish peroxidase-conjugated secondary antibody, followed by incubation for 2 hours. Then, protein expression was detected using ECL detection reagent (Amersham). The protein bands were scanned using a GS-800 calibrated imaging densitometer (Bio-Rad, Hercules, CA, USA) and the density of each band using a Quantity one 1-D analysis program (Bio-Rad). Was analyzed. Data are expressed as the ratio of target protein to α-tubulin in treated and control samples.

2-5. In vitro Modify( IVF )

For IVF, B6D2F1/CrljOri hybrid female mice (10-12 weeks old) were purchased from OrientBio® (Gapyeong, Gyeonggi-do, Korea). These mice were intraperitoneally injected with pregnant mare serum gonadotrophin (5 IU) and human chorionic gonadotrophin (hCG, 5 IU) every 48 hours to induce hyperovulation (Rahman et al., 2015, 2018; Ryu et al., 2017b ). About 15 hours after hCG injection, cumulus-oocyte complexes were collected, transferred to BM containing 10% fetal bovine serum in mineral oil, and incubated for 1 hour in an environment of 37° C. and 5% CO ₂ . In addition, about 30 oocytes were used in each treatment group collected from at least 3 mice, and BM containing 0.4% BSA was used for the fertilization ability of sperm collected from male mice and exposed to BPA/antioxidants. , 1×10 ⁶ /mL sperm were fertilized with cultured oocytes. As previously described (Rahman et al., 2015, 2018), oocytes were cultured for 6 hours in an environment of 37° C. and 5% CO ₂ . After fertilization, the normal conjugate was transferred to BM containing 0.4% BSA. Fertilization rate was evaluated by counting the number of 2-cell embryos (negative percentage) 24 hours after fertilization. All two-cell embryos were transferred back to BM containing 0.4% BSA and the number of blastocysts was counted after 5 days.

Ratio of cleavage or blastocyst (%)=

2-6. Statistical analysis

Data were analyzed using the One-way Analysis of ANOVA (ANOVA) of SPSS version 23.0 (Chicago, IL, USA), and Tukey's HSD test was performed to confirm significant differences between the different treatment groups. P<0.05 was considered to represent a statistically significant difference, and the values presented herein represent mean±standard error (SEM).

Experimental example One. On BPA Exposed sperm motility Effect of antioxidants on parameters

Sperm motility is an essential predictor of male fertility and optimal motility, and motor dynamics are the criterion for successful fertilization (Suarez, 2008; Rahman et al., 2017b). Therefore, first, it was evaluated whether the antioxidant protects the motility parameter in sperm exposed to BPA by the method of Example 2-1.

As a result, as reported in the previous study (Rahman et al., 2015, 2016), it was found that 100 μM of BPA significantly reduced the proportion of motile sperm compared to the control group (P <0.05). However, in BPA-exposed sperm supplemented with antioxidants, motility was significantly higher than in BPA alone treatment group (P <0.05). In addition, in the GSH and Vit E combination exposure group, the reduced motion characteristics were completely recovered, but not in the Vit C group (P<0.05). Unlike overall motility, minor changes were observed in curved velocity (VCL), average path (VAP), and linear velocity (VSL). However, other epidemiologic parameters of sperm did not show a significant difference between the control and treatment groups.

Table 1 below shows the changes in sperm kinetics parameters by treatment with BPA and antioxidants.

In Table 1, MOT (motility) is motility, HYT (hyperactivated motility) is overactive motility, VCL (curvilinear velocity) is a curved velocity, VSL (straight-line velocity) is a linear velocity, VAP (average path velocity) is an average path, LIN (linearity) is linearity, BCF (beat cross frequency) is frequency, WOB (wobble) is shake, DNC (dance) is movement, DNM (dance mean) is movement average, ALH (mean amplitude of head lateral displacement) is head Mean amplitude of lateral displacement, and data represent mean ±SEM of 6 repetitions (n=3 mice/repeat).

Parameters Control BPA BPA + GSH BPA + Vit C BPA + Vit E MOT (%) 81.54 ± 0.88 ^A 51.39 ± 1.91 ^B 64.51 ± 1.64 ^C 66.55 ± 2.22 ^C 67.40 ± 3.11 ^C HYP (%) 9.66 ± 0.87 8.35 ± 0.37 11.31 ± 1.07 8.55 ± 1.23 8.93 ± 1.36 VCL (μm/s) 113.90 ± 3.61 ^A,B 113.38 ± 3.22 ^A,B 122.81 ± 3.30 ^B 102.93 ± 2.20 ^A 115.23 ± 3.69 ^B VSL (μm/s) 42.82 ± 1.10 ^A 39.45 ± 2.00 ^A,B 43.64 ± 0.98 ^A 37.09 ± 1.24 ^B 41.32 ± 1.2 ^A,B VAP (μm/s) 54.89 ± 2.27 ^A 51.22 ± 1.34 ^A,B 55.44 ± 1.29 ^A 46.76 ± 1.40 ^B 53.91 ± 1.05 ^A LIN (%) 38.09 ± 1.54 36.57 ± 1.25 35.93 ± 0.37 37.91 ± 1.19 35.88 ± 0.33 BCF 13.57 ± 0.54 12.91 ± 0.37 12.38 ± 0.23 13.57 ± 0.31 13.18 ± 0.24 WOB 48.36 ± 0.88 46.08 ± 0.39 45.55 ± 0.69 47.73 ± 0.75 46.89 ± 0.85 DNC 578.37 ± 15.15 ^A 444.78 ± 13.48 ^B 640.2 ± 11.12 ^A 421.41 ± 9.86 ^B 585.74 ± 8.2 ^A DNM (μm) 13.37 ± 1.23 13.24 ± 0.59 14.63 ± 0.48 11.35 ± 0.51 13.13 ± 0.42 ALH (μm) 4.96 ± 0.32 4.78 ± 0.14 5.24 ± 0.13 4.29 ± 0.12 5.07 ± 0.13

Experimental example 2. On BPA Acquisition of fertility of exposed sperm Effect of antioxidants on the aspect

For successful fertilization, the mammalian sperm must undergo complex biochemical changes in the female reproductive organs, collectively known as'capacitation', and cause acrosomal exocytosis (Rahman et al., 2017a, 2017b; Visconti, 2009). Therefore, the change in fertility acquisition pattern of BPA-exposed sperm treated with BPA alone and antioxidants by the method of Example 2-2 was evaluated.

As a result, as shown in Figure 3a, the acrosome-reacted sperm count (AR pattern) was significantly increased in the group treated with BPA alone in vitro , and sperm exposed to BPA were additionally supplemented with GSH and Vit E. At this time, the number of AR sperm was similar to that of the control group, unlike Vit C (P <0.05). However, as shown in Figs. 3b and 3c, there was no significant difference in the number of sperm with fertilization ability (Fig. 3b) and sperm without fertilization ability (Fig. 3c) in BPA alone or in BPA and antioxidant treatment groups.

Experimental example 3. Protein Kinase-A ( PKA ) Active and BPA Tyrosine in exposed sperm Effect of antioxidants on phosphorylation

In particular, protein phosphorylation for tyrosine residues and PKA activity is important for the acrosome reaction of mammalian sperm for motility activation, fertility acquisition and fertilization (Rahman et al., 2016, 2017a, 2017b; Visconti, 2009). Therefore, the effect of BPA alone or BPA and antioxidant-treated groups on sperm parameters as described above was confirmed by the Western blotting analysis of Example 2-4 and shown in FIG. 4A, which was quantified and shown in FIG. 4B. .

As a result, as in the previous study (Rahman et al., 2015; Wan et al., 2016), it was confirmed that PKA activity in sperm was particularly increased in the 16 kDa band of the BPA-only treatment group (Fig. 4a), BPA It was confirmed that PKA activity was similar to that of the control group in the and antioxidant-treated groups (Fig. 4b).

In addition, as shown in Fig. 4c, a similar pattern of change was also observed in the protein tyrosine phosphorylation level of the 60 kDa band of BPA alone or BPA and antioxidant treatment group, as shown in Fig. 4d, and 23 kDa band than the control sperm in all treatment groups. Showed significantly higher protein tyrosine phosphorylation in (P <0.05)

Experimental example 4. On BPA In the cells of the exposed sperm ROS And protein tyrosine To nitration Effects of antioxidants on

Low levels of ROS are essential for regulation of sperm function. However, when the production of these latent oxygen metabolites increases, it causes spontaneous tyrosine nitration of proteins and causes oxidative stress on cells (Rahman et al., 2018, Herrero et al., 2001). Therefore, in order to elucidate the mechanism of the stress response of sperm according to different treatment conditions, the intracellular ROS was measured by the method of Example 2-3 and the tyrosine nitration level was confirmed.

As a result, as shown in FIG. 5A, significantly higher ROS levels were observed in the BPA-treated group than in the control group and other groups (P<0.05), and at the same time, the oxidative stress induced by the increased ROS in the BPA and antioxidant-treated groups was observed. It was confirmed that it was completely recovered.

On the other hand, as a result of Western blotting by the method of Example 2-4, as shown in FIG. 5B, both the BPA-only treatment group and the BPA and antioxidant treatment groups showed higher protein tyrosine nitration (16 kDa molecular weight) than the control group. (P<0.05), BPA+GSH and BPA+Vit C treatment group showed significantly lower tyrosine nitration compared to BPA alone treatment group, but BPA+Vit E treatment group showed BPA only. There was no significant difference compared to the treatment group.

The DCFDA method of Example 2-3 used for the ROS detection assay was based on 2',7' dichlorofluorescin diacetate dye, which is a cell permeable reagent capable of measuring intracellular hydroxyl, peroxyl and other ROS activities, Tyrosine nitration is induced by peroxynitrite (ONOO-) (Saura et al., 2014). Thus, although antioxidants reduced total ROS in BPA-exposed sperm, sperm could not completely overcome ONOO-mediated stress.

Experimental example 5. On BPA Fertilization of exposed sperm and early embryonic development Effect of antioxidants on ability

Under normal circumstances, progesterone, secreted by the amorphous egg cell, promotes the acrosome response in the sperm before fertilization. Therefore, if the acrosome reaction occurs in the sperm prior to meeting the oocyte, these sperm have fertility (Rahman et al., 2015). Early acrosome responses are associated with altered mitochondrial activity and increased chromatin decondensation associated with oxidative stress (Chaveiro et al., 2006; Wongtawan et al., 2006).

Thus, the ability of sperm for fertilization and early embryo development was investigated using in vitro fertilization (IVF) analysis by the method of Example 2-5. In particular, antioxidants can protect sperm fertility after exposure to BPA. I checked if it can.

As a result, as shown in Fig. 6, it was confirmed that GSH and Vit E can recover the fertilization and early embryo-generating ability of sperm exposed to BPA (P<0.05). Vit C slightly increased fertilization and embryonic development by sperm exposed to BPA, but the actual increase was not statistically significant.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the examples and experimental examples described above are illustrative in all respects and are not limiting.

Claims (7)

A composition for alleviating reproductive toxicity by bisphenol A (Bisphenol-A; BPA) containing glutathione as an active ingredient.
delete The method of claim 1,
The glutathione (glutathione) is characterized in that the concentration is 1mM to 10mM, bisphenol A (Bisphenol-A; BPA) for reproductive toxicity alleviation composition.
delete delete The method of claim 1,
The reproductive toxicity by the bisphenol A is any one or more of the generation of reactive oxygen species, the activation of protein kinase A (PKA), the nitration of protein tyrosine, the decrease in the acquisition of sperm fertility, or the decrease in the early embryonic development ability of sperm. Characterized in that, bisphenol A (Bisphenol-A; BPA) for reproductive toxicity alleviation composition.
A food composition for preventing or improving reproductive toxicity by bisphenol-A (BPA), containing glutathione as an active ingredient.

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