Method and composition for evaluating influence on melanin production

Abstract

To provide means of evaluating the in vitro influence on melanin production.SOLUTION: A method of evaluating the influence of a test substance on melanin production is provided, comprising measuring the degree of endoplasmic reticulum stress in melanin-producing cells in contact with the test substance.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to a method for evaluating an effect on melanin production, a composition for evaluating an effect on melanin production, and the like. [Background technology]

[0002] Melanin is a brown pigment produced by melanin-producing cells and is distributed in the skin, hair, eyeballs, etc. Various ingredients that inhibit melanin production have been proposed as skin-whitening ingredients.

[0003] On the other hand, vitiligo is a condition in which the skin loses pigment partially due to functional modification of melanin-producing cells (e.g., a decrease in the amount of melanin produced by melanin-producing cells or a decrease in the number of melanin-producing cells), resulting in the appearance of white patchy areas. For example, there have been nearly 20,000 reported cases (in humans) of vitiligo caused by external exposure to rhododenol (RD), a type of skin-whitening ingredient (Non-Patent Document 1).

[0004] In addition, several to several dozen cases (in humans) have been reported in which white spots developed due to external exposure to raspberry ketone (RK) (Non-Patent Document 4), 4-tert-butylpyrocatechol (4TBC) (Non-Patent Document 5), 4-tert-amylphenol (4TAP) (Non-Patent Document 6), 4-tert-butylphenol (4TBP) (Non-Patent Document 7), hydroquinone (HQ) (Non-Patent Document 8), and 4-tert-octylphenol (4TOP) (Non-Patent Document 9). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Inoue S, Katayama I, Suzuki T, Tanemura A, Ito S, Abe Y, Sumikawa Y, Yoshikawa M, Suzuki K, Yagami A, Masui Y, Ito A, Matsunaga K vitiligo. J Dermatol. 48, 969–9

Outdoor Tool2

Outdoor Tools3

Outdoor Tools 4

Direct Environment 5

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Summary of the Invention

[0006] Currently, risk assessment of vitiligo caused by chemicals such as rhododenol mainly uses in vivo evaluation systems using animal models (Non-Patent Document 2), and there is a demand for the development of in vitro risk assessment technology that can quickly and low-costly evaluate the vitiligo risk of various chemicals. Furthermore, if it were possible to evaluate the effects of various chemicals on melanin production in vitro, this would be useful as a means of discovering skin-whitening ingredients. An object of the present disclosure is to provide a means for evaluating the effect on melanin production in melanin-producing cells in vitro. Another object is to provide a composition for evaluating the effect on melanin production in melanin-producing cells. Another object of the present disclosure is to provide a means for evaluating vitiligo risk in vitro. Another object is to provide a composition for evaluating vitiligo risk. [Means for solving the problem]

[0007] The present inventors discovered that chemical substances that can induce vitiligo in humans induce endoplasmic reticulum stress, and have made further improvements.

[0008] The present disclosure includes, for example, the subject matter described in the following sections: Section 1. (II) A method for evaluating the effect of a test substance on melanin production, comprising a step of measuring the degree of endoplasmic reticulum stress in melanin-producing cells contacted with the test substance. Section 2. Prior to step (II), (I) The method according to Item 1, comprising a step of contacting a test substance with melanin-producing cells. Section 3. Item 3. The method according to Item 1 or 2, wherein the evaluation of the effect of the test substance on melanin production is a vitiligo risk evaluation of the test substance. Section 4. The degree of endoplasmic reticulum stress is measured by detecting an endoplasmic reticulum stress marker, The method according to any one of items 1 to 3, wherein the endoplasmic reticulum stress marker is at least one selected from the group consisting of mature XBP1, phosphorylated IRE1α, MANF, TXNIP, phosphorylated JNK1, phosphorylated PERK, phosphorylated eIF2α, ATF4, CHOP / GADD153, NOXA, phosphorylated NRF2, HMOX1, NQO1, ATF6f, BiP, PDIA4, HYOU1, SEL1L, EDEM1, HERPUD1, and fragments thereof. Section 5. Item 4. The method according to any one of Items 1 to 3, wherein the level of endoplasmic reticulum stress is measured by detecting mature XBP1 or a fragment thereof. Section 6. Item 6. The method according to any one of Items 1 to 5, wherein the melanin-producing cells are melanocytes. Section 7. Item 7. The method according to any one of items 1 to 6, wherein the vitiligo is non-photosensitive vitiligo. Section 8. A composition for evaluating an effect on melanin production, for use in the method according to any one of items 1 to 7, comprising an agent for detecting an endoplasmic reticulum stress marker. Section 9. A composition for assessing a risk of vitiligo, for use in the method according to any one of items 3 to 7, comprising an agent for detecting an endoplasmic reticulum stress marker. Effect of the Invention

[0009] A means for evaluating an effect on melanin production in vitro is provided. Also, a composition for evaluating an effect on melanin production is provided. Also, a means for evaluating a vitiligo risk in vitro is provided. Also, a composition for evaluating a vitiligo risk is provided. [Brief description of the drawings]

[0010] [Figure 1]The chemical formulas of seven chemicals that can induce vitiligo (rhododenol (RD), raspberry ketone (RK), 4-tert-butylpyrocatechol (4TBC), 4-tert-amylphenol (4TAP), 4-tert-butylphenol (4TBP), hydroquinone (HQ), and 4-tert-octylphenol (4TOP)) and three chemicals that can induce photosensitive vitiligo (losartan potassium (LP), hydrochlorothiazide (HCT), and flutamide (FT)) are shown. [Diagram 2] 1 shows the results of measuring the efficiency of tyrosinase knockdown using siRNA. [Diagram 3] The results of in vitro tyrosinase-dependent cell death after chemical treatment are shown. The cell viability of melanocyte cell lines 24 hours after treatment with different concentrations of chemicals is shown relative to the vehicle control (mean ± standard deviation, n = 3). Note that melanocytes were transfected with siRNA (siControl: ○, siTYR: △) 24 hours before chemical treatment. **P<0.01. [Figure 4] 1 shows the results of measuring reactive oxygen species (ROS) production in vitro after treatment with RD. [Diagram 5] The results of in vitro ROS measurements after chemical treatment are shown. The relative ROS production of each chemical was quantified by total intensity of CellROX Green normalized by cell number compared to vehicle-treated cells (n=3). **P<0.01, *P<0.05. NS indicates not significant. [Figure 6](A) Schematic diagram of endoplasmic reticulum (ER) stress sensor plasmid. Expression of XBP1, which contains a splicing site located upstream of the termination codon fused to Venus, is controlled under the CAGGS promoter / enhancer. Under normal conditions, the sensor mRNA is not spliced, and therefore the Venus protein is not translated. Under ER stress conditions, the spliced ​​mRNA undergoes a frameshift, and the Venus protein is translated. (B) Representative photographs after induction of ER stress by tunicamycin or RD treatment are shown. [Figure 7] ER stress response after 24-hour treatment of melanocyte cell lines with chemicals at IC50 is shown relative to vehicle control (mean ± SD, n=3). **P<0.01, *P<0.05. NS indicates not significant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Each embodiment included in the present disclosure will be described in further detail below. The present disclosure encompasses a method for evaluating the effect of a test substance on melanin production, comprising the step of (II) measuring the degree of endoplasmic reticulum stress in melanin-producing cells contacted with the test substance. In the present specification, the method may be referred to as the "evaluation method of the present disclosure." In addition, in the present specification, the step may be referred to as step (II).

[0012] The melanin producing cells are not particularly limited as long as the effects of the present disclosure are achieved. Examples include melanocytes, melanoma cells, etc. Among these, melanocytes are preferred. Furthermore, the melanin-producing cells may be primary cultured cells, subcultured cells, or established cell lines. The species of organism from which the melanin-producing cells are derived is preferably, for example, a mammal. More specifically, examples include humans; rodents such as mice, rats, guinea pigs, and hamsters; dogs, cats, rabbits, cows, monkeys, and pigs. Among these, humans or rodents are preferred, and humans or mice are more preferred. Furthermore, the melanin-producing cells may be cells differentiated from stem cells such as iPS cells and ES cells.

[0013] The test substance is not particularly limited and may be, for example, a compound, a complex, a composition, etc. The compound may be, for example, a low molecular weight compound, a nucleic acid (e.g., DNA, RNA, etc.), a protein (e.g., an antibody or a part thereof, etc.), a sugar chain, a polymer, etc. The complex may be, for example, a compound formed by non-covalent association of the same or different compounds as described above. The composition may be an extract obtained from an organism (e.g., an animal, a plant, a microorganism, etc.), or may be a combination of two or more compounds. The test substance may be, for example, one that induces tyrosinase-dependent cell death or one that does not induce tyrosinase-dependent cell death. Also, the test substance may be, for example, one that induces the production of reactive oxygen species or one that does not induce the production of reactive oxygen species.

[0014] Endoplasmic reticulum stress refers to a state in which proteins that have not been folded normally (structurally abnormal proteins) accumulate in the endoplasmic reticulum.

[0015] Examples of methods for measuring the level of endoplasmic reticulum stress include a method for detecting an endoplasmic reticulum stress marker and a method for detecting protein aggregation in the endoplasmic reticulum. These can be used alone or in combination of two or more. Among them, the method for detecting an endoplasmic reticulum stress marker is preferred from the viewpoints of simplicity, accuracy, and the like.

[0016] Examples of endoplasmic reticulum stress sensor proteins that have been reported include inositol requiring protein 1α (IRE1α), PRKR-like endoplasmic reticulum kinase (PERK), and Activating Transcription Factor 6 (ATF6) (Non-Patent Document 3).

[0017] IRE1α, a type I endoplasmic reticulum membrane protein, senses the accumulation of structurally abnormal proteins in the endoplasmic reticulum lumen through its luminal domain and is activated by phosphorylation of serine 724 or serine 729. Activated IRE1α cleaves X-box binding protein 1 (XBP1u) mRNA at two specific sites with its cytoplasmic ribonuclease domain to initiate a splicing reaction. In this reaction, a 26-base intron is excised from the open-reading frame (ORF) of XBP1u mRNA, and then the 5' and 3' fragments are ligated by tRNA ligase RTCB. As a result, mature XBP1 (spliced ​​XBP1: XBP1s) in which a frameshift has occurred in the sequence after the splicing site is translated. Examples of target genes of mature XBP1 include mesencephalic astrocyte-derived neurotrophic factor (MANF) and thioredoxin-interacting protein (TXNIP). In addition, IRE1α causes activation (phosphorylation) of c-Jun N-terminal kinase (JNK1) by interacting with TRAF2.

[0018] PERK, a type I transmembrane protein, has a stress-sensing domain in the endoplasmic reticulum lumen and a protein kinase domain in the cytoplasm. When PERK dimerizes and / or oligomerizes in stressed cells, it becomes activated (phosphorylated), which phosphorylates the α-subunit of eukaryotic translation initiation factor 2 α (eIF2α), suppressing global translation and reducing protein load in the endoplasmic reticulum lumen. Although cap-dependent translation is suppressed, transcription factors such as ATF4 are selectively induced in response to phosphorylation of eIF2α. Target genes of ATF4 include, for example, genes related to endoplasmic reticulum stress-induced apoptosis, such as CHOP / GADD153 and NOXA. Activation of PERK also induces phosphorylation of the transcription factor NRF2, whose target genes include heme oxygenase 1 (HMOX1) and NAD(P)H quinone dehydrogenase 1 (NQO1).

[0019] ATF6 is a 90 kDa type II single-pass transmembrane protein, and two mutants, ATF6α and ATF6β, exist in mammalian cells. Under endoplasmic reticulum stress, ATF6 translocates from the endoplasmic reticulum to the Golgi apparatus, where it is cleaved by two serine proteases (S1P and S2P) to generate a 50 kDa active basic leucine zipper (bZip) transcription factor (ATF6f). Target genes of ATF6f include BiP, Protein Disulfide Isomerase Family A Member 4 (PDIA4), Hypoxia up-regulated protein 1 (HYOU1), Suppressor / Enhancer Lin12-like (SEL1L), ER degradation-enhancing alpha-mannosidase-like protein 1 (EDEM1), and Homocysteine ​​Inducible ER Protein With Ubiquitin Like Domain 1 (HERPUD1).

[0020] The endoplasmic reticulum stress marker used for measuring the degree of endoplasmic reticulum stress is not particularly limited as long as it can measure the degree of endoplasmic reticulum stress. For example, mature XBP1, phosphorylated IRE1α, MANF, TXNIP, phosphorylated JNK1, phosphorylated PERK, phosphorylated eIF2α, ATF4, CHOP / GADD153, NOXA, phosphorylated NRF2, HMOX1, NQO1, ATF6f, BiP, PDIA4, HYOU1, SEL1L, EDEM1, HERPUD1, and fragments thereof can be mentioned. As the endoplasmic reticulum stress marker, one kind can be used alone or two or more kinds can be used in combination. Among them, it is preferable to use mature XBP1 or a fragment thereof. These are endoplasmic reticulum stress markers whose amount and / or concentration increases during the endoplasmic reticulum stress response. If the amount and / or concentration of the endoplasmic reticulum stress marker increases, it can be determined that the degree of endoplasmic reticulum stress has increased.

[0021] The method for detecting the endoplasmic reticulum stress marker is not particularly limited, and may be appropriately selected depending on the type of the endoplasmic reticulum stress marker, such as a method for directly and specifically detecting a part or all of the endoplasmic reticulum stress marker, a method for indirectly detecting the endoplasmic reticulum stress marker, etc. More specifically, examples of the method include a reporter assay method, an immunological measurement method using an antibody that specifically recognizes the endoplasmic reticulum stress marker, an RT-PCR method, a Northern blot method, etc.

[0022] Amino acid sequence information of endoplasmic reticulum stress markers and their coding sequence information can be easily obtained from various databases such as NCBI.

[0023] The reporter assay method can be performed according to known methods, for example, by constructing a reporter vector in which the transcriptional regulatory region of a gene encoding an endoplasmic reticulum stress marker is inserted upstream of a gene encoding a reporter protein, and introducing the reporter vector into melanin-producing cells. The reporter protein is not particularly limited as long as it is a luminescent (color-producing) protein that emits light (color) upon reaction with a specific substrate, or a fluorescent protein that emits fluorescence upon excitation light. Examples of the reporter protein include fluorescent proteins such as Venus, mCherry, and GFP; luciferase; β-galactosidase, chloramphenicol acetyltransferase, and β-glucuronidase. The type of vector is not particularly limited, and examples thereof include plasmid vectors such as animal cell expression plasmids; and viral vectors such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, and Sendai virus. The method for introducing the expression vector into the melanin-producing cells is not particularly limited, and any known method can be used. In addition, the gene encoding the reporter protein may be fused to a gene encoding an endoplasmic reticulum stress marker.

[0024] For example, when a protein encoded by a gene spliced ​​by endoplasmic reticulum stress, such as mature XBP1, is used as an endoplasmic reticulum stress marker, the endoplasmic reticulum stress marker can be detected by detecting the presence or absence of splicing. For example, in the reporter assay method, an expression vector containing a polynucleotide in which a gene encoding an endoplasmic reticulum stress marker and a gene encoding a reporter protein are fused can be used so that the reporter protein is expressed only when the gene encoding the endoplasmic reticulum stress marker is spliced ​​(in this specification, the expression vector may be referred to as an "endoplasmic reticulum stress sensor expression vector"). More specifically, the degree of endoplasmic reticulum stress can be measured by introducing an endoplasmic reticulum stress sensor expression vector containing a polynucleotide in which a gene encoding a reporter protein is fused downstream of the splicing site of the gene encoding the endoplasmic reticulum stress marker into a melanin-producing cell. That is, under a non-endoplasmic reticulum stress response, the gene encoding the endoplasmic reticulum stress marker is not spliced, so that frameshift does not occur and the reporter protein is not expressed. On the other hand, under an endoplasmic reticulum stress response, the gene encoding the endoplasmic reticulum stress marker is spliced, so that frameshift occurs and the reporter protein is expressed. Alternatively, an expression vector containing a polynucleotide in which a gene encoding an endoplasmic reticulum stress marker is fused to a gene encoding a reporter protein can be used, so that the reporter protein is not expressed only when the gene encoding the endoplasmic reticulum stress marker is spliced.

[0025] In addition, the degree of endoplasmic reticulum stress can also be measured by the above-mentioned method, for example, by using a primer, probe, antibody, etc. that can detect the presence or absence of splicing. Examples of primers, probes, or antibodies that can detect the presence or absence of splicing include primers that amplify across a splicing site, primers and probes that hybridize to a splicing site, and antibodies that bind to a splicing site.

[0026] Examples of immunological measurement methods include Western blotting, ELISA, sandwich ELISA, EIA, RIA, and immunohistochemical staining.

[0027] Methods for detecting protein aggregation in the endoplasmic reticulum include, for example, thioflavin T staining. During the endoplasmic reticulum stress response, protein aggregation increases. If protein aggregation increases, it can be determined that the degree of endoplasmic reticulum stress has increased.

[0028] The evaluation method of the present disclosure may include, prior to step (II), a step (I) of contacting a test substance with melanin-producing cells.

[0029] The method for contacting the test substance with the melanin-producing cells is not particularly limited. For example, the melanin-producing cells and the test substance may be added to an appropriate solvent, or the test substance may be added to the melanin-producing cells. The suitable solvent is not particularly limited, and examples thereof include water, liquid medium for cell culture, and the like. The concentration of the test substance can be appropriately set depending on the type of the test substance. The temperature during contact is not particularly limited and can be, for example, about 25 to 40°C, or about 30 to 40°C, etc. The contact time is not particularly limited and can be, for example, about 1 minute to 48 hours, or about 10 minutes to 24 hours. During the contact, for example, stirring or shaking can be performed as necessary.

[0030] According to the evaluation method of the present disclosure, a test substance that increases the level of endoplasmic reticulum stress when contacted with the test substance compared to when not contacted with the test substance can be evaluated as being capable of suppressing melanin production. Examples of suppression of melanin production include a decrease in the amount of melanin produced by melanin-producing cells, a decrease in the melanin-producing cells themselves, etc.

[0031] The evaluation method of the present disclosure can be used, for example, for evaluating the risk of vitiligo of a test substance. In other words, in the evaluation method of the present disclosure, the evaluation of the effect of a test substance on melanin production may be, for example, evaluation of the risk of vitiligo of the test substance.

[0032] It has been reported that the above-mentioned chemical substances such as rhododenol (RD, RK, 4TBC, 4TAP, 4TBP, HQ, and 4TOP) induce vitiligo when exposed to them alone. On the other hand, it is known that there are chemical substances (LP, HCT, and FT) that do not induce vitiligo by themselves but do induce vitiligo when combined with light such as sunlight (in this specification, such vitiligo may be referred to as "photosensitive vitiligo"). Examples of vitiligo that is the subject of the present disclosure include vitiligo induced by external exposure to a chemical substance alone (without light) (in this specification, such vitiligo may be referred to as "non-photosensitive vitiligo"), photosensitive vitiligo, etc. Among them, non-photosensitive vitiligo is preferred.

[0033] According to the evaluation method of the present disclosure, a test substance that increases the level of endoplasmic reticulum stress when contacted with the test substance compared to when not contacted with the test substance can be evaluated as having a risk of vitiligo.

[0034] The present disclosure also includes a composition for evaluating the effect on melanin production, comprising an agent for detecting an endoplasmic reticulum stress marker. In this specification, the composition may be referred to as the "composition for evaluation of the present disclosure."

[0035] The endoplasmic reticulum stress marker detection agent is not particularly limited as long as it can detect the endoplasmic reticulum stress marker. For example, an endoplasmic reticulum stress sensor expression vector (more specifically, an expression vector containing a polynucleotide in which a gene encoding an endoplasmic reticulum stress marker and a gene encoding a reporter protein are fused so that the reporter protein is expressed only when endoplasmic reticulum stress is received and splicing occurs); a reporter vector capable of confirming the expression level of an endoplasmic reticulum stress marker (for example, a reporter vector in which a transcriptional regulatory region of a gene encoding an endoplasmic reticulum stress marker is inserted upstream of a gene encoding a reporter protein); a primer or probe that specifically recognizes a gene encoding an endoplasmic reticulum stress marker; an antibody that specifically recognizes an endoplasmic reticulum stress marker; and the like. These can be used alone or in combination of two or more kinds.

[0036] The agent for detecting an endoplasmic reticulum stress marker can be prepared by a method known in the art depending on the type.

[0037] In the evaluation composition of the present disclosure, the content of the endoplasmic reticulum stress marker detection agent is not particularly limited and can be set appropriately depending on the type of endoplasmic reticulum stress marker detection agent.

[0038] The evaluation composition of the present disclosure includes an endoplasmic reticulum stress marker detection agent and may further include other components. Examples of the other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, surfactants, antioxidants, preservatives, coating agents, colorants, fragrances, pH adjusters, etc. These components may be used alone or in combination of two or more.

[0039] The evaluation composition of the present disclosure can be prepared by a conventional method by combining an endoplasmic reticulum stress marker detection agent and, if necessary, other components.

[0040] The evaluation composition of the present disclosure can be used in the evaluation method of the present disclosure. In addition, the evaluation composition of the present disclosure can be used, for example, for evaluating the risk of vitiligo of a test substance. In other words, the evaluation composition of the present disclosure can be said to be, for example, a composition for evaluating the risk of vitiligo of a test substance.

[0041] In addition, in this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure includes any combination of the constituent elements described in this specification.

[0042] In addition, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter included in the present disclosure. In other words, the present disclosure includes all subject matter consisting of all combinations of each combinable characteristic described in this specification. EXAMPLES

[0043] The contents of the present disclosure will be specifically explained using the following experimental examples. However, the present disclosure is not limited to these in any way. In the following, unless otherwise specified, the experiments are performed under atmospheric pressure and room temperature conditions. Furthermore, unless otherwise specified, "%" means "% by mass".

[0044] As shown in Table 1, seven chemical substances were selected from human case reports as chemical substances that can induce vitiligo: rhododenol (RD) (Non-Patent Document 1), raspberry ketone (RK) (Non-Patent Document 4), 4-tert-butylpyrocatechol (4TBC) (Non-Patent Document 5), 4-tert-amylphenol (4TAP) (Non-Patent Document 6), 4-tert-butylphenol (4TBP) (Non-Patent Document 7), hydroquinone (HQ) (Non-Patent Document 8), and 4-tert-octylphenol (4TOP) (Non-Patent Document 9). On the other hand, three chemical substances were selected as chemical substances that do not induce vitiligo in the absence of light such as sunlight (i.e., can induce photosensitive vitiligo), unlike the above-mentioned chemical substances: losartan potassium (LP) (Non-Patent Document 10), hydrochlorothiazide (HCT) (Non-Patent Document 10), and flutamide (FT) (Non-Patent Document 11) (the structural formulas of each chemical substance are shown in Figure 1). The following experiments were performed under conditions without light induction. Therefore, in the following experiments, the first seven chemicals were used as positive controls and the latter three chemicals were used as negative controls.

[0045] RD (Wako) and 4TBC (Sigma-Aldrich) were dissolved in 70% ethanol and used in the following experiments. RK (Tokyo Chemical Industry Co., Ltd.), 4TAP (Sigma-Aldrich), HQ (Sigma-Aldrich), 4TOP (Sigma-Aldrich), 4TBP (Sigma-Aldrich), LP (Tokyo Chemical Industry Co., Ltd.), HCT (Tokyo Chemical Industry Co., Ltd.), and FT (Tokyo Chemical Industry Co., Ltd.) were dissolved in dimethyl sulfoxide (DMSO) and used in the following experiments.

[0046] [Table 1]

[0047] Previous in vitro studies have reported that causative factors of vitiligo induced by RD include tyrosinase-dependent cell death (Non-Patent Document 12), reactive oxygen production (Non-Patent Document 13), and endoplasmic reticulum stress (Non-Patent Document 12). The present inventors measured tyrosinase-dependent cell death, reactive oxygen production, and endoplasmic reticulum stress for the above-mentioned 10 chemical substances.

[0048] Analysis of cultured melanocytes Immortalized mouse normal melanocyte cell line (melan-a), provided by Dr. Bennett, was cultured in RPMI-1640 (Wako) supplemented with 10% FBS (Gibco), 1% penicillin-streptomycin-amphotericin B suspension (Wako), and 200 nM 12-O-tetradecanoylphorbol-13-acetate (Wako) at 37°C and 5% CO2 (Thang et al. 2017. Deltex-3-like (DTX3L) stimulates metastasis of melanoma through FAK / PI3K / AKT but not MEK / ERK pathway. Oncotarget. 6, 14290-14200., Omata et al. 2018. A disadvantageous effect of adsorption of barium by melanin on transforming activity. Chemosphere. 210, 384-391.). After 24 hours of adherent culture, melanocytes were exposed to various concentrations of chemicals for 24 hours. Then, cells were stained with DAPI (Wako), cell numbers were counted, and IC 50 was determined (Table 1).

[0049] Tyrosinase-dependent cell death Transfection of siRNA was performed using Lipofectamine RNAi MAX (Thermo Fisher Scientific) according to the manufacturer's protocol. The efficiency of tyrosinase knockdown was evaluated by qPCR (Figure 2). IC 50 The cells were treated with 10 chemicals ranging from high to low concentrations for 24 hours, and the cells were counted to measure tyrosinase-dependent cell death.

[0050] Reactive oxygen production The effects of 10 chemicals on ROS production were examined in a melanocyte cell line. ROS was quantified 30 min after chemical treatment using CellROX Green reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. The IC 50 Relative ROS production in cells was quantified by total CellROX Green intensity normalized to cell number and compared to vehicle-treated cells.

[0051] Endoplasmic reticulum stress Quantification of endoplasmic reticulum stress response was performed by transfecting melanocytes with the endoplasmic reticulum stress sensor plasmid (XBP1-venus) provided by Dr. Miura (Iwawaki et al. 2004. A transgenic mouse model for monitoring endoplasmic reticulum stress. 2004. Nat Med. 10, 98-102.) and Caggs-mCherry plasmid (Rodrigo et al. 2015. Planar cell polarity-mediated induction of neural stem cell expansion during axolotl spinal cord regeneration. Elife. 4, e10230.) as a transfection control (Figure 6A). Transfection of plasmid DNA was performed using TransIT-X2 (Takara) according to the manufacturer's protocol. 24 hours after transfection, the medium was changed to a chemical (IC 50 )-containing medium. 24 hours after chemical treatment, cells were fixed and the relative ER stress response of each chemical was quantified as the percentage of Venus-positive cells among mCherry-positive cells compared to vehicle treatment.

[0052] statistical analysis Differences between the chemical-exposed and non-exposed groups were analyzed using the Mann-Whitney U test (Gu et al. 2022. Decreased hearing levels at frequencies for understanding speech in tannery workers exposed to a high level of trivalent chromium in Bangladesh. Chemosphere. 306, 135571). Statistical analysis was performed using SPSS version 27.0 (IBM Corp).

[0053] As shown in Figure 3, RD and RK, which has a similar chemical structure to RD, induced tyrosinase-dependent cell death, but the other eight chemicals did not induce tyrosinase-dependent cell death.

[0054] As shown in Figures 4 and 5, among the seven chemicals that can induce vitiligo in humans, RD, RK, 4TBC, and 4TBP showed increased ROS production, while 4TAP, HQ, and 4TOP were comparable to the vehicle control. In addition, the ROS production levels of the three negative control chemicals were all comparable to the vehicle control.

[0055] As shown in Figure 6B, VENUS-positive melanocytes were confirmed to induce ER stress response when exposed to tunicamycin or RD, which have been reported to induce ER stress response. As shown in Figure 7, all seven chemicals that can induce vitiligo in humans induced ER stress response. Meanwhile, all three negative control chemicals showed ER stress response at the same level as the vehicle control.

[0056] Reported causative factors of vitiligo induced by RD include tyrosinase-dependent cell death (Non-Patent Document 12), production of reactive oxygen species (Non-Patent Document 13), and endoplasmic reticulum stress (Non-Patent Document 12). It has been found that the risk of vitiligo can be evaluated by using the degree of endoplasmic reticulum stress as an indicator.

Claims

1. (II) A method for evaluating the effect of a test substance on melanin production, comprising a step of measuring the degree of endoplasmic reticulum stress in melanin-producing cells contacted with the test substance.

2. Prior to step (II), The method according to claim 1, comprising the step of (I) contacting a test substance with melanin-producing cells.

3. The method according to claim 1, wherein the evaluation of the effect of the test substance on melanin production is a vitiligo risk assessment of the test substance.

4. The degree of endoplasmic reticulum stress is measured by detecting an endoplasmic reticulum stress marker, The method according to any one of claims 1 to 3, wherein the endoplasmic reticulum stress marker is at least one selected from the group consisting of mature XBP1, phosphorylated IRE1α, MANF, TXNIP, phosphorylated JNK1, phosphorylated PERK, phosphorylated eIF2α, ATF4, CHOP / GADD153, NOXA, phosphorylated NRF2, HMOX1, NQO1, ATF6f, BiP, PDIA4, HYOU1, SEL1L, EDEM1, HERPUD1, and fragments thereof.

5. The method according to any one of claims 1 to 3, wherein the degree of endoplasmic reticulum stress is measured by detecting mature XBP1 or a fragment thereof.

6. The method according to any one of claims 1 to 3, wherein the melanin producing cells are melanocytes.

7. The method according to any one of claims 1 to 3, wherein the vitiligo is non-photosensitive vitiligo.

8. A composition for evaluating an effect on melanin production, for use in the method according to claim 1 or 2, comprising an agent for detecting an endoplasmic reticulum stress marker.

9. A composition for assessing the risk of vitiligo for use in the method described in claim 3, comprising an agent for detecting an endoplasmic reticulum stress marker.

Images