Method for predicting harmfulness of inhalable chemical substance

Abstract

To provide a method for predicting the harmfulness of an inhalable chemical substance using an alveolar macrophage model experimental system.SOLUTION: A method according to the present invention includes the following steps of: (1) administering a test substance to a lung cell-derived cell, and obtaining a list (LT) of relative expression levels of mRNA of each gene of a discriminant index gene group in the lung cell-derived cell to which the test substance has been administered, from RNA obtained by culturing and recovering the lung cell-derived cell; and (2) predicting the level of harmfulness of the test substance by discriminant analysis on the basis of the LTt and lists (LPC) and (LNC) of relative mRNA expression levels of each gene in the discriminant index gene group in cells derived from lung cells administered with a positive control substance and a negative control substance; wherein the discriminant index gene group includes 18 types of genes of Ahcyl1 and the like. The present invention also provides pairs of primers comprising nucleotide sequences such as SEQ ID NO: 1 and / or 2 for use in the methods of the present invention.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for predicting the harmfulness of inhalable chemicals based on the gene expression levels of cultured cells administered with the inhalable chemicals, and more specifically, to a method for predicting the harmfulness based on the increase / decrease patterns of gene expression in immortalized mouse alveolar macrophages administered with an inhalable inorganic chemical. [Background technology]

[0002] Evaluating the hazards of inhalable chemicals is extremely important in order to prevent health problems from occurring at the workplace. To date, the hazards of inhalable chemicals have been evaluated using animal testing, but due to the high cost, the need for expertise, the time it takes to make a judgment, and the issue of animal welfare, animal testing should be limited. Therefore, a new screening test using cultured cells is needed to determine whether animal testing is necessary.

[0003] Among inhalable chemicals, insoluble particles of inorganic chemicals are inhaled into the respiratory system and adhere to the airway mucosa or alveoli, and do not return to the airflow. This state is called deposition. Most of the particulate matter deposited in the airways and alveolar cavities is eliminated by a clearance mechanism mainly based on the mucociliary epithelial system. The alveolar region is the site where gas exchange takes place, and the alveoli are composed of flat type I alveolar epithelial cells and cuboidal type II alveolar epithelial cells scattered between them, and alveolar macrophages with phagocytic ability are present in the alveolar walls. Microparticles with a particle diameter of 1 μm are said to reach the alveoli, but most are expelled by exhalation. Particles deposited on the alveolar walls are phagocytosed by alveolar macrophages and transported to the lower respiratory tract region, but some are transported to the lymphatic system via the interstitium.

[0004] There are already several publications that use cultured cells to evaluate the harmfulness of chemicals. For example, Non-Patent Document 1 reports predicting the renal toxicity of chemicals using primary human kidney cells. Non-Patent Document 2 reports predicting the developmental toxicity of chemicals using primary fetal rat brain nerve cells. However, there are no reports predicting the lung toxicity of inhaled chemicals using lung-derived cells. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Ramm S, et al. Toxicol Sci. 2019 May 1;169(1):54-69. [Non-Patent Document 2] Kotake Y, Abstract of the 95th Annual Meeting of the Japanese Pharmacological Society (2022), 1-S09-4 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a method for predicting the harmfulness of inhaled chemicals using a model experimental system of alveolar macrophages, which play a key role in the clearance of inhaled chemicals that have reached the alveolar space. A further object of the present invention is to provide a method for predicting the harmfulness of inhaled chemicals based on gene expression patterns in a model experimental system of alveolar macrophages administered with the inhaled chemical. [Means for solving the problem]

[0007] The present inventors used immortalized mouse alveolar macrophages as a model experimental system for alveolar macrophages to analyze gene expression patterns in immortalized mouse alveolar macrophages exposed to inhalable inorganic chemicals in culture. However, the gene expression patterns vary greatly for each individual inhalable inorganic chemical. As a result of extensive research, the present inventors succeeded in identifying a group of genes (hereinafter referred to as a "discrimination indicator gene group") whose expression levels change in common with harmful inhalable inorganic chemicals, thereby completing the present invention.

[0008] That is, the present invention relates to the following. [1] 1. A method for predicting the toxicity of a test substance that is an inhalable chemical, comprising: (1) A test substance is administered to a cell derived from a lung cell, and the cell derived from the lung cell is cultured and then recovered. From the RNA obtained, a list of relative expression levels of mRNA of each gene in the group of discriminant genes in the cell derived from the lung cell to which the test substance is administered (L T ), (2) Said L T and a list of the relative expression levels of mRNA for each gene in the discriminant gene group in cells derived from lung cells administered with a positive control substance (L PC ) and a list of the relative expression levels of mRNA for each gene in the discriminant gene group in lung cells derived from negative control substances (L NC ) A step of predicting the level of toxicity of a test substance by discriminant analysis based on the above. method. [2] The method according to [1], wherein the group of discriminant indicator genes consists of or includes Ahcyl1, Asah1, Atxn10, Clpb, Cog6, Crot, Cuta, Fam173a, Fam96a, Nusap1, Oat, Pdia6, Rps6ka1, Rsl1d1, Sc5d, Timeless, Xrcc6 and Yars. [3] The method according to [1] or [2], wherein the test substance is an inhalable inorganic chemical. [4] The method according to any one of [1] to [3], wherein the harmful effect of the test substance is lung damage. [5] Said L PC teeth, (a) administering a positive control substance to a cell derived from a lung cell, culturing the cell derived from the lung cell, and recovering the RNA obtained from the RNA, the absolute expression level of each gene of the discriminant gene group in the cell derived from the lung cell administered with the positive control substance; and (b-1) the absolute expression level of the mRNA of the internal control gene in cells derived from the lung cells administered with the positive control substance, and / or (b-2) the absolute expression level of mRNA of each gene in the group of discriminant indicator genes in cells derived from lung cells administered with only the solvent of the positive control substance; Said L NC teeth, (c) administering a negative control substance to a cell derived from a lung cell, culturing the cell derived from the lung cell, and recovering the RNA obtained from the cell, the absolute expression level of each gene in the group of discriminant genes in the cell derived from the lung cell administered with the negative control substance; and (d-1) the absolute expression level of the mRNA of the internal control gene in cells derived from the lung cells administered with the negative control substance, and / or (d-2) the absolute expression level of mRNA of each gene in the group of discriminant indicator genes in cells derived from lung cells administered with only the solvent of the negative control substance, Said L T teeth, (e) administering a test substance to a cell derived from a lung cell, culturing the cell derived from the lung cell, and recovering the RNA obtained from the cell, the absolute expression level of each gene of the discriminant gene group in the cell derived from the lung cell administered with the test substance; and (f-1) the absolute expression level of the mRNA of an internal control gene in cells derived from lung cells administered with the test substance, and / or (f-2) the absolute expression level of mRNA of each gene in the group of discriminant indicator genes in cells derived from lung cells administered with only the solvent for the test substance; The method according to any one of [1] to [4]. [6] The method according to any one of [1] to [5], wherein the expression level of mRNA of each gene of the discriminant indicator gene group in a cell derived from a lung cell administered with the test substance, a positive control substance, and / or a negative control substance is quantified using a pair of primers, a probe, or a single-stranded circular template comprising or consisting of a nucleotide sequence of SEQ ID NO: 1 and / or 2, 3 and / or 4, 5 and / or 6, 7 and / or 8, 9 and / or 10, 11 and / or 12, 13 and / or 14, 15 and / or 16, 17 and / or 18, 19 and / or 20, 21 and / or 22, 23 and / or 24, 25 and / or 26, 27 and / or 28, 29 and / or 30, 31 and / or 32, 33 and / or 34, and 35 and / or 36, or a nucleotide sequence complementary to the nucleotide sequence. [7] The method according to [6], wherein the expression level of mRNA of each gene of the discriminant indicator gene group in cells derived from lung cells administered the test substance, positive control substance, and / or negative control substance is quantified by PCR using primer pairs of SEQ ID NOs: 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, and 35 and 36. [8] The method according to any one of [1] to [7], wherein the cell derived from a lung cell is an alveolar macrophage. [9] The method according to [8], wherein the alveolar macrophages are mouse MH-S cells.

[10] The method according to any one of [5] to [9], wherein the internal standard gene is selected from the group consisting of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin, hypoxanthine phosphoribosyltransferase 1 (HPRT1), β2 microglobulin (B2M) and hydroxymethylbilane synthase (HMBS).

[11] A primer pair, a probe, or a single-stranded circular template for use in the method according to any one of [1] to

[10] , comprising or consisting of a nucleotide sequence of SEQ ID NO: 1 and / or 2, 3 and / or 4, 5 and / or 6, 7 and / or 8, 9 and / or 10, 11 and / or 12, 13 and / or 14, 15 and / or 16, 17 and / or 18, 19 and / or 20, 21 and / or 22, 23 and / or 24, 25 and / or 26, 27 and / or 28, 29 and / or 30, 31 and / or 32, 33 and / or 34, and 35 and / or 36, or a nucleotide sequence complementary to said nucleotide sequence.

[12] A kit for use in the method according to any one of [1] to

[10] , comprising a pair of primers, a probe, or a single-stranded circular template, the kit comprising or consisting of the nucleotide sequence of SEQ ID NO: 1 and / or 2, 3 and / or 4, 5 and / or 6, 7 and / or 8, 9 and / or 10, 11 and / or 12, 13 and / or 14, 15 and / or 16, 17 and / or 18, 19 and / or 20, 21 and / or 22, 23 and / or 24, 25 and / or 26, 27 and / or 28, 29 and / or 30, 31 and / or 32, 33 and / or 34, and 35 and / or 36, or a nucleotide sequence complementary to the nucleotide sequence. Effect of the Invention

[0009] According to the present invention, it is possible to provide a method for predicting the lung hazard of a test substance, which is an inhalable inorganic chemical substance, using cultured cells instead of animal testing, and also to provide a method for predicting the lung hazard of a large number of test substances in a short period of time. [Brief description of the drawings]

[0010] [Figure 1] Figures 1-1 to 1-5 show the calculation formulas used in the discriminant analysis of Table 5. [Diagram 2]Figures 2-1 to 2-4 show the calculation formulas used in the discriminant analysis of Table 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] One embodiment of the present invention is a method for predicting the level of toxicity of a test substance that is an inhalable chemical substance, comprising the steps of: (1) administering the test substance to a cell derived from a lung cell, culturing the cell, and recovering the RNA obtained to obtain a list of relative expression levels of mRNA of each gene in a group of discriminant indicator genes in the cell administered with the test substance (L T ), and (2) obtaining the L T and a list of the relative expression levels of mRNA for each gene in the discriminant gene group in cells administered with the positive control substance (L PC ) and a list of the relative expression levels of mRNA for each gene in the discriminant gene group in cells administered with the negative control substance (L NC ) and predicting whether the test substance is highly harmful or not by discriminant analysis based on the above.

[0012] In this specification, inhalable chemicals refer to chemicals that can be deposited in the lungs. According to the permissible concentration set by the Japan Society for Occupational Health, inhalable chemicals are defined as having a particle diameter of 7.07 μm or less. Particles larger than this size, even if exposed at high concentrations, are deposited in small amounts in the lungs, resulting in poor reactions in the lungs and, as a result, the biological effects are underestimated. For example, it is known that the alveolar deposition rate of inhalable chemical particles with a particle diameter of 0.5 to 5 μm is around 10%, while the alveolar deposition rate of inhalable chemical particles with a particle diameter of 0.1 μm or less is several times that rate. Inhalable chemicals include organic chemicals and inorganic chemicals.

[0013] In conventional inhalation exposure tests, the toxicity of test substances, which are inhalable chemical substances, has been evaluated based on the presence or absence of the onset of lung inflammation, fibrosis, tumors, etc., and the severity of the pathology. When foreign substances such as chemicals enter the lungs, alveolar macrophages and other cells recognize and phagocytose the foreign substances and attempt to excrete them from the lungs. If the foreign substances cannot be excreted promptly, the alveolar macrophages release liquid components such as cytokines and chemokines, which mobilize and accumulate inflammatory cells such as neutrophils and macrophages, forming a pathology known as inflammation. If this inflammation continues, the cells that protect the lungs (alveoli) themselves are damaged, and in compensation, fibers such as collagen fibers are excessively deposited, which is the formation of fibrosis. Furthermore, in the presence of inflammation and fibrosis, repeated stimulation of epithelial cells causes damage to genes in the nucleus, transforming them into tumor cells, resulting in tumor formation (Morimoto et al., Int. J. Mol. Sci., 17, E-165 (2016)). In other words, inflammation persists and the epithelium is not repaired, leading to the next step of fibrosis and tumor formation.

[0014] Therefore, the present invention provides a method for predicting the harmfulness of a test substance, which is an inhalable chemical substance, based on the response of cells derived from lung cells exposed to the test substance, preferably on changes in gene expression patterns.

[0015] Positive control substances refer to inhalable chemicals that have been demonstrated to have high lung toxicity in inhalation exposure tests, which are the gold standard tests for lung toxicity assessment, including, but not limited to, NiO nano, CeO2 nano, and crocidolite. Negative control substances refer to inhalable chemicals that have been demonstrated to have low lung toxicity in inhalation exposure tests, including, but not limited to, P90 TiO2 nano, XT3, and XT4.

[0016] As the lung cell-derived cells used in the method of the present invention, alveolar macrophages, particularly immortalized alveolar macrophages, are preferred. As a medium for culturing these cells, a mammalian cell medium containing 5-15% fetal bovine serum (FBS) and / or serum of other animal species, or a serum-free medium supplemented with 0.5-5 mM L-glutamine, 0.01 mM-0.1 mM 2-mercaptoethanol, etc., particularly a macrophage medium, for example, Ham's F12K medium supplemented with 15% fetal bovine serum (FBS) and 2 mM L-glutamine, or RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 0.05 mM 2-mercaptoethanol, can be used.

[0017] The discriminant gene group is determined based on the change in the pattern of increase or decrease in gene expression by performing mRNA expression analysis in cells derived from lung cells administered with a positive control substance or a negative control substance, as follows: For mRNA expression analysis, comprehensive analysis such as microarray analysis using 10,000 or more probes or RNA-Seq (RNA sequence) analysis using a next-generation sequencer is used.

[0018] When genes of the discriminant gene group are selected by microarray analysis, the expression level is obtained as the intensity of the fluorescent signal due to hybridization between fluorescently labeled mRNA and each solid-phase probe, or its relative value. For example, the Global Normalization method explained in Mestdagh P. et al. (Genome Biology 10, R64. (2009) is used for data normalization in the case of microarray analysis. Specifically, a Global Normalization value normalized so that the median of the sum of signal intensities is 25 may be used. In the examples of this specification, the median-normalized relative value of the expression level (E MedIn RNA-Seq analysis, expression levels are obtained as read counts. Data normalization in RNA-Seq analysis involves normalizing the total mRNA read counts in cells administered a positive control substance or a negative control substance to the same value (e.g., 1 million). The relative expression levels of mRNA are determined based on the results of these normalizations.

[0019] In both the case of microarray analysis and RNA-Seq analysis, the conditions for selecting genes of the discriminant indicator gene group include, but are not limited to, that for each gene, the lowest relative value of the expression level of mRNA in cells administered with multiple positive control substances is higher than the highest relative value of the expression level of mRNA in cells administered with multiple negative control substances, and that there is a clear statistically significant difference (e.g., a significant difference under conditions of t-test, two-sided test, and unequal variances).

[0020] In the present embodiment, the E Med The 18 genes listed in Table 3, which had a p value of 50 or more and a significant difference of p<0.05, were determined as genes of the discriminant indicator gene group. These genes are Ahcyl1, Asah1, Atxn10, Clpb, Cog6, Crot, Cuta, Fam173a, Fam96a, Nusap1, Oat, Pdia6, Rps6ka1, Rsl1d1, Sc5d, Timeless, Xrcc6, and Yars. However, since the genes of the discriminant indicator gene group may differ depending on the cells to which the test substance is administered, the genes of the discriminant indicator gene group of the present invention are not limited to these.

[0021] In the present invention, the relative expression level of mRNA of each gene of the discriminant gene group in the cells administered with the test substance is normalized for the expression levels of different genes, and the ratio of the normalized relative value of the expression level of each mRNA to the normalized relative value of the internal standard gene is called the internal standard normalized relative value (E Int) is calculated. Here, the internal standard gene refers to a gene whose expression level does not change or whose expression level does not differ significantly between the administration of a positive control substance or a negative control substance and the administration of nothing in cells derived from lung cells to which a test substance is administered. Specific examples of internal standard genes include, but are not limited to, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin, hypoxanthine phosphoribosyltransferase 1 (HPRT1), β2 microglobulin (B2M), and hydroxymethylbilane synthase (HMBS). These genes are well known to those skilled in the art.

[0022] In the present invention, the solvent used when administering a test substance, a positive control substance, or a negative control substance to cells includes, but is not limited to, sterile distilled water or physiological saline.

[0023] In the present specification, the ratio of the internal standard normalized relative value of the expression level of the mRNA of each gene in the cells administered with each test substance to the internal standard normalized relative value of the expression level of the mRNA of each gene in the cells administered with only the solvent not containing each test substance is defined as the final relative value of the expression level of the mRNA of each gene in the cells administered with each test substance (E Rel )

[0024] In the present invention, the E of each gene of the discriminant gene group obtained from the cells administered with the test substance Rel The set of values ​​is converted into a list of relative expression levels of mRNA of each gene in the discriminant gene group in cells administered with the test substance (L T Similarly, the E of each gene in the group of discriminant indicator genes obtained from cells administered with a positive control substance is Rel The set of values ​​is then converted into a list of the relative expression levels of mRNA for each gene in the discriminant gene group in cells administered with a positive control substance (L PC ) The E of each gene in the group of discriminant indicator genes obtained from cells administered with a negative control substance was Rel The set of values ​​is then converted into a list of the relative expression levels of mRNA for each gene in the discriminant gene group in cells administered with the negative control substance (L NC ) is called.T , L PC and L NC may be in any format that can be recognized by discriminant analysis software running on a computer.

[0025] The discriminant analysis used in the step of predicting the level of toxicity of a test substance of the present invention is Fisher's linear discriminant analysis method based on the assumption that the distribution of all classes has the same covariance structure. In this specification, L is calculated using array analysis and quantitative real-time PCR. T , L PC and L NC The discriminant analysis of the present invention is not limited by the OS or statistical processing program of the computer, provided that Fisher's linear discriminant analysis method can be performed on the numerical data of the relative expression level of mRNA for each gene in the discriminant indicator gene group.

[0026] One embodiment of the present invention is a method for predicting the level of toxicity of a test substance that is an inhalable chemical substance, wherein the discriminant indicator gene group is a group consisting of or including Ahcyl1, Asah1, Atxn10, Clpb, Cog6, Crot, Cuta, Fam173a, Fam96a, Nusap1, Oat, Pdia6, Rps6ka1, Rsl1d1, Sc5d, Timeless, Xrcc6, and Yars. The structures and functions of the genes in the discriminant indicator gene group of this embodiment are described in Table 3.

[0027] One embodiment of the invention is a method for predicting the high or low harmfulness of a test substance which is an inhalable chemical, wherein the test substance is an inhalable inorganic chemical.

[0028] One embodiment of the invention is a method for predicting the high or low toxicity of a test substance that is an inhalable chemical, wherein the test substance is an inhalable organic chemical.

[0029] As used herein, respirable organic chemicals include, but are not limited to, powdered cross-linked water-soluble acrylic polymer chemicals and polyvinyl chloride. In this specification, the inhalable inorganic chemicals include oxides of magnesium, titanium, nickel, cobalt, cerium, and the like (e.g., P90 TiO2nano (Yoshiura, Y. et al. J Nanopart Res. 2015;17(6):241.), NiO nano (Oyabu, T. et al. Int J Mol Sci. 2017 Dec 19;18(12):2757.), CeO2nano (Morimoto, Y. et al. J Nanopart Res. 2015;17(11):442.)), talc, amorphous silica, asbestos (e.g., chrysotile, crocidolite, Nagatomo, H. et al. Inhal Toxicol. 2005 May;17(6):293-6.), fine powder of printer toner (e.g., XT3 (Tomonaga, T. et al. BioMed Research International, (2017) Volume 2017, Article ID 4245309, 12 pages; Tomonaga, T. et al. J Occup Health. 2020;62:e12146.), XT4, BY5, BY6, BY7 and BY9). The literature in parentheses immediately following each chemical is an example of a literature that examined the pulmonary toxicity of each test substance.

[0030] One embodiment of the present invention is a method for predicting the toxicity of a test substance that is an inhalable chemical, comprising the steps of: Said L PC teeth, (a) administering a positive control substance to a cell derived from a lung cell, culturing the lung cell, and recovering the RNA obtained, the absolute expression level of each gene of the discriminant gene group in the cell administered with the positive control substance; and (b-1) the absolute expression level of the mRNA of the internal control gene in the cells administered with the positive control substance, and / or (b-2) the absolute expression level of mRNA of each gene in the group of discriminant indicator genes in cells administered with only the solvent of the positive control substance; Said L NC teeth, (c) administering a negative control substance to a cell derived from a lung cell, culturing the lung cell, and recovering the RNA obtained, the absolute expression level of each gene in the group of discriminant genes in the cell treated with the negative control substance; and (d-1) the absolute expression level of the mRNA of the internal control gene in the cells treated with the negative control substance, and / or (d-2) the absolute expression level of mRNA of each gene in the group of discriminant indicator genes in cells administered with only the solvent of the negative control substance, Said L T teeth, (e) administering a test substance to a cell derived from a lung cell, culturing the lung cell, and recovering the RNA obtained, the absolute expression level of each gene in the discriminant gene group in the cell treated with the test substance; and (f-1) the absolute expression level of the mRNA of the internal control gene in the cells treated with the test substance, and / or (f-2) The method is based on the absolute expression level of mRNA of each gene in the group of discriminant indicator genes in cells administered with only the solvent of the test substance.

[0031] As used herein, the term "absolute expression level of mRNA" refers to the measured value of mRNA, for example, the measured value of a specific mRNA signal in microarray analysis. The absolute expression level is normalized or otherwise processed for calculation to obtain a relative value of the expression level.

[0032] One embodiment of the present invention is a method for predicting the toxicity of a test substance that is an inhalable chemical, comprising the steps of: The method includes quantifying the expression level of mRNA of each gene of the discriminant indicator gene group in cells administered with the test substance, positive control substance, and / or negative control substance of the present invention using a pair of primers, a probe, or a single-stranded circular template comprising or consisting of the nucleotide sequence of SEQ ID NO: 1 and / or 2, 3 and / or 4, 5 and / or 6, 7 and / or 8, 9 and / or 10, 11 and / or 12, 13 and / or 14, 15 and / or 16, 17 and / or 18, 19 and / or 20, 21 and / or 22, 23 and / or 24, 25 and / or 26, 27 and / or 28, 29 and / or 30, 31 and / or 32, 33 and / or 34, and 35 and / or 36, or a nucleotide sequence complementary to said nucleotide sequence.

[0033] One embodiment of the present invention is a method for predicting the toxicity of a test substance that is an inhalable chemical, comprising the steps of: The method comprises quantifying the mRNA expression level of each gene in the discriminant indicator gene group in cells administered with the test substance, positive control substance and / or negative control substance of the present invention by PCR using primer pairs of SEQ ID NOs: 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, and 35 and 36.

[0034] L of the present invention T , L PC and L NCTo obtain the above, the array analysis method including the microarray analysis and the RNA-Seq analysis method described above may be used, but when the number of genes in the indicator gene group is small, an expression level analysis method specific to each of the genes can be used. For example, the method includes, but is not limited to, quantitative real-time PCR and other PCR methods, rolling circle amplification including LAMP (Loop-mediated Isothermal Amplification), and array analysis using a dedicated array in which genes in the indicator gene group are immobilized on a solid phase. As a primer pair used in PCR, a probe used in array analysis, or a single-stranded circular template used in rolling circle amplification, an oligonucleotide or polynucleotide that includes or consists of the nucleotide sequence of SEQ ID NO: 1 and / or 2, 3 and / or 4, 5 and / or 6, 7 and / or 8, 9 and / or 10, 11 and / or 12, 13 and / or 14, 15 and / or 16, 17 and / or 18, 19 and / or 20, 21 and / or 22, 23 and / or 24, 25 and / or 26, 27 and / or 28, 29 and / or 30, 31 and / or 32, 33 and / or 34, and 35 and / or 36, or a nucleotide sequence complementary to said nucleotide sequence, can be used.

[0035] One embodiment of the present invention is a method for predicting whether a test substance, which is an inhalable chemical substance, is highly harmful, wherein the cells derived from the lung cells of the present invention are alveolar macrophages.

[0036] One embodiment of the present invention is a method for predicting the level of toxicity of a test substance, which is an inhalable chemical substance, wherein the alveolar macrophage of the present invention is a mouse MH-S cell.

[0037] In the present specification, the alveolar macrophage includes immortalized alveolar macrophages as well as alveolar macrophages or primary cultured cells thereof collected from a living body. As the immortalized alveolar macrophage, established cultured cells such as NR8383 cells (rat) and MH-S cells (mouse) can be used, and it can also be newly prepared by infection with SV40 or other immortalizing, transforming or tumorigenic viruses or by transfection with oncogenes.

[0038] One embodiment of the present invention is a method for predicting the level of harmfulness of a test substance that is an inhalable chemical, wherein the internal standard gene is selected from the group consisting of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin, hypoxanthine phosphoribosyltransferase 1 (HPRT1), β2 microglobulin (B2M) and hydroxymethylbilane synthase (HMBS).

[0039] One embodiment of the invention is a pair of primers, a probe or a single stranded circular template comprising or consisting of the nucleotide sequence of SEQ ID NO: 1 and / or 2, 3 and / or 4, 5 and / or 6, 7 and / or 8, 9 and / or 10, 11 and / or 12, 13 and / or 14, 15 and / or 16, 17 and / or 18, 19 and / or 20, 21 and / or 22, 23 and / or 24, 25 and / or 26, 27 and / or 28, 29 and / or 30, 31 and / or 32, 33 and / or 34, and 35 and / or 36, or a nucleotide sequence complementary thereto, for use in the method of the invention.

[0040] One embodiment of the invention is a kit comprising a pair of primers, a probe or a single stranded circular template comprising or consisting of the nucleotide sequence of SEQ ID NO: 1 and / or 2, 3 and / or 4, 5 and / or 6, 7 and / or 8, 9 and / or 10, 11 and / or 12, 13 and / or 14, 15 and / or 16, 17 and / or 18, 19 and / or 20, 21 and / or 22, 23 and / or 24, 25 and / or 26, 27 and / or 28, 29 and / or 30, 31 and / or 32, 33 and / or 34, and 35 and / or 36, or a nucleotide sequence complementary to said nucleotide sequence, for use in the method of the invention.

[0041] The kit of the present invention is useful for easily carrying out the method of the present invention. The probes in the kit of the present invention may be immobilized on a solid phase. The solid phase may be a slide glass on which all the probes are immobilized at regular intervals, and may have a size, shape, and material that can be measured by a fluorescent signal measuring device for array analysis.

[0042] In this specification, the conjunction "about" used to modify a numerical value means a numerical range of 90% or more and 110% or less of the numerical value. For example, when the dose of a test substance is "about 100 μg," the dose is within a numerical range including 90 μg and 110 μg, which are the lower and upper limits, respectively.

[0043] All documents mentioned herein are incorporated by reference in their entirety.

[0044] The following examples of the present invention are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. The present invention can be modified, for example, by adding, deleting, and replacing the constituent elements of the present invention, without departing from the spirit of the present invention. EXAMPLES

[0045] 1. Test substances used The test materials used in this example were TiO2nano (MT-150AW), TiO2nano (P90), print toner (XT3), NiO nano (US3355), CeO2nano (Wako), silica, chrysotile, and crocidolite.

[0046] The test substances and solvents used in this example and the results of the lung damage evaluation in rats are shown in Table 1 below.

[0047] [Table 1]

[0048] 2.Analysis method 1 x 10 immortalized rat alveolar macrophage NR8383 cells 6 The cells were seeded in a 35 mm culture dish containing 2 mL of Ham's F12K medium supplemented with 15% fetal bovine serum (FBS) and 2 mM L-glutamine, and 24 hours later, 50 or 100 μg of each test substance was administered. The purified total RNA was used for mRNA microarray analysis (3D-Gene, TORAY INDUSTRIES, INC., number of rat probes installed: mRNA 20,174). For each test substance, the background value was subtracted from the raw data of each sample based on the measured expression level of each gene (E), and normalized to a median of 25 to obtain the median normalized relative expression level (E Med ) was decided.

[0049] 3. Determination of genes in the discriminant gene group The ratio of the internal standard normalized relative value of the expression level of each gene in cells administered with each test substance to the internal standard normalized relative value of the expression level of each gene in cells administered with only the solvent not containing each test substance was defined as the final relative value of the expression level of each gene in cells administered with each test substance (E Rel ) was decided.

[0050] The number of genes for which values ​​of mRNA expression levels were obtained in cells administered all test substances was 12,942. Rel The lowest value of E for test substances with low lung toxicity Rel There were 447 genes with higher values ​​than the highest value of E Rel The lowest value of E for test substances with high lung toxicity Rel No gene had a higher value than the highest value.

[0051] The statistical analysis software JMP Pro 15's hierarchical cluster analysis function was used to further narrow down the results and determine the E of test substances with high lung toxicity. Rel The lowest value of E for test substances with low lung toxicity Rel There were 13 genes whose E values ​​were 1.5 times higher than the highest value of the test substance with high lung damage. Rel However, there were 168 genes with more than 100 genes for all test substances. And there were four genes common to these 13 genes and the 168 genes: Abca1, Ddit3, Hist1h2bh, and Lsp1.

[0052] In the hierarchical cluster analysis using the statistical analysis software JMP pro 15, the analysis results of test substances with high and low lung toxicity based on the expression levels of the above four types of genes were equivalent to the analysis results based on the expression levels of the above 447 types of genes. EXAMPLES

[0053] 1. Test substances used The test materials used in this example are P90 TiO2 nano, XT3, XT4, BY5, BY6, BY7, BY9, NiO nano, CeO2 nano and Crocidolite.

[0054] The test substances and solvents used in this example and the results of the lung damage evaluation in rats are shown in Table 1 below.

[0055] [Table 2]

[0056] 2.Analysis method As in Example 1, 3 × 10 MH-S cells (immortalized mouse alveolar macrophages) were 5 The cells were seeded in a 35 mm culture dish containing 2 mL of RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 0.05 mM 2-mercaptoethanol, and 24 hours later, 100 μg of each test substance was administered. After 24 hours, the cells were harvested and total RNA was purified.

[0057] As in Example 1, purified total RNA was used to perform mRNA microarray analysis (3D-Gene, TORAY INDUSTRIES, INC., number of mouse probes installed: 23,474). For each test substance, the measured expression level (E) of each gene was normalized so that the median of the sum of E was 25 according to Zhao Y. et al. (BMC Bioinformatics, 6:28 (2005)) and Mestdagh P. et al. (Genome Biology 10, R64 (2009)), to obtain the median-normalized relative expression level (E Med ) was decided.

[0058] 3. Determination of genes in the discriminant gene group The ratio of the median normalized relative value of the expression level of each mRNA to the median normalized relative value of the expression level of the internal standard gene (GAPDH) was defined as the internal standard normalized relative value (E Int ) was calculated. Furthermore, the ratio of the internal standard normalized relative value of the expression level of the mRNA of each gene in the cells administered with each test substance to the internal standard normalized relative value of the expression level of the mRNA of each gene in the cells administered with only the solvent not containing each test substance was defined as the final relative value of the expression level of the mRNA of each gene in the cells administered with each test substance (E Rel ) was decided.

[0059] Next, the Effect of Each Gene on ... Rel Based on the above, the significant differences were analyzed using t-test, two-tailed test, and unequal variance. Med There were 18 genes with a value of 50 or more and a significant difference of p<0.05, as shown in Table 2 below.

[0060] [Table 3]

[0061] Hereinafter, in this specification, the combination of the 18 types of genes in Table 3 will be referred to as a group of discriminant indicator genes.

[0062] 4. E of genes in the discriminant gene group in microarray Med Discriminant analysis using E of genes in the discriminant index gene group Rel The following are shown in Tables 4-1 to 4-3. Rel was normalized by correcting the median to 25, and was calculated as a relative value to the control group (expression level of cells administered only with a solvent not containing a test substance).

[0063] [Table 4-1]

[0064] [Table 4-2]

[0065] [Table 4-3]

[0066] The E of the genes in the discriminant gene group in Table 4-1 or Table 4-3Rel Based on the data and the results of the degree of lung damage of each test substance, a discriminant analysis was performed using JMP (registered trademark) pro15. The results are shown in Table 5. The calculation formula used for the discriminant analysis in Table 5 is shown in Figures 1-1 to 1-5.

[0067] [Table 5]

[0068] 5. Discriminant analysis using the measured expression levels of genes in the discriminant indicator gene group by quantitative real-time PCR Since the group of discriminant indicator genes includes 18 types of genes, the expression levels can be quantified by PCR. In this embodiment, a quantitative real-time PCR method (hereinafter referred to as "RT-qPCR method") was used as the PCR method. The primer pairs for quantifying the expression levels of each gene in the group of discriminant indicator genes and the internal standard GAPDH gene are shown in Table 6 and the sequence table below.

[0069] [Table 6]

[0070] The expression levels of genes in the discriminant indicator gene group in RNA recovered and purified from MH-S cells cultured for 24 hours after administration of the test substances P90 TiO2nano, XT3, XT4, BY5, BY6, BY7, BY9, NiO nano, CeO2nano and Crocidolite or their solvents were quantified by real-time quantitative PCR (ΔΔCT method using SYBR Green: internal standard was GAPDH) using each primer pair listed in Table 6. The E of the relative expression level of mRNA of each gene in the discriminant indicator gene group in cells administered with the test substance is expressed as the relative ratio of the expression level of genes in the discriminant indicator gene group in RNA in MH-S cells administered with each test substance to the expression level of genes in RNA in MH-S cells administered with only the solvent of each test substance. Rel The following are shown in Tables 7-1 to 7-3.Rel was normalized by correcting for GAPDH and calculated as a relative value to the control group (expression level of cells administered with only a solvent not containing the test substance).

[0071] [Table 7-1]

[0072] [Table 7-2]

[0073] [Table 7-3]

[0074] The results of discriminant analysis performed based on the lists of relative expression levels in Tables 7-1 to 7-3 are shown in Table 8. The calculation formulas used in the discriminant analysis in Table 8 are shown in Figures 2-1 to 2-4.

[0075] [Table 8]

[0076] As described above, when the lung injury assessment of each test substance, "high" or "low," was input as the actual measured value into the discriminant analysis program based on the list of relative expression levels of genes in the discriminant gene group obtained by real-time quantitative PCR, the program output a predicted value of "high" or "low," which all coincided with the actual measured value. In other words, the method of the present invention succeeded in appropriately predicting lung injury. [Industrial Applicability]

[0077] The method of the present invention for predicting the level of harmfulness of a test substance, which is an inhalable chemical substance, can predict harmfulness based on the expression of genes of a group of discriminant indicator genes in cultured cells to which the test substance has been administered, thereby reducing unnecessary animal testing and improving animal welfare.

Claims

1. 1. A method for predicting the toxicity of a test substance that is an inhalable chemical, comprising: (1) A test substance is administered to a cell derived from a lung cell, and the cell derived from the lung cell is cultured and then recovered. From the RNA obtained, a list of relative expression levels of mRNA of each gene in the group of discriminant genes in the cell derived from the lung cell to which the test substance is administered (L Ｔ ) and (2) Said L Ｔ and a list of the relative expression levels of mRNA of each gene in the group of discriminant indicator genes in cells derived from lung cells administered with a positive control substance (L ＰＣ ) and a list of the relative expression levels of mRNA of each gene in the group of discriminant indicator genes in cells derived from lung cells administered with a negative control substance (L ＮＣ ) and predicting whether the test substance is highly harmful or not by discriminant analysis based on the above. method.

2. The method according to claim 1, wherein the group of discriminant indicator genes consists of or includes Ahcyl1, Asah1, Atxn10, Clpb, Cog6, Crot, Cuta, Fam173a, Fam96a, Nusap1, Oat, Pdia6, Rps6ka1, Rsl1d1, Sc5d, Timeless, Xrcc6 and Yars.

3. 3. The method of claim 1 or 2, wherein the test substance is a respirable inorganic chemical.

4. The method according to any one of claims 1 to 3, wherein the harmful effect of the test substance is lung damage.

5. Said L ＰＣ teeth, (a) administering a positive control substance to a cell derived from a lung cell, culturing the cell derived from the lung cell, and recovering the RNA obtained from the RNA, the absolute expression level of each gene of the discriminant gene group in the cell derived from the lung cell administered with the positive control substance; and (b-1) the absolute expression level of the mRNA of the internal control gene in cells derived from lung cells administered with the positive control substance, and / or (b-2) the absolute expression level of mRNA of each gene in the group of discriminant indicator genes in cells administered with only the solvent of the positive control substance; Said L ＮＣ teeth, (c) administering a negative control substance to a cell derived from a lung cell, culturing the cell derived from the lung cell, and recovering the RNA obtained from the RNA, the absolute expression level of each gene of the group of discriminant genes in the cell derived from the lung cell administered with the negative control substance; and (d-1) the absolute expression level of the mRNA of the internal control gene in cells derived from lung cells administered with the negative control substance, and / or (d-2) the absolute expression level of mRNA of each gene in the group of discriminant indicator genes in cells derived from lung cells administered with only the solvent of the negative control substance; Said L Ｔ teeth, (e) administering a test substance to a cell derived from a lung cell, culturing the cell derived from the lung cell, and recovering the RNA obtained from the RNA, the absolute expression level of each gene of the discriminant gene group in the cell derived from the lung cell administered with the test substance; and (f-1) the absolute expression level of the mRNA of an internal standard gene in a cell derived from a lung cell of the test substance, and / or (f-2) the absolute expression level of mRNA of each gene in the group of discriminant indicator genes in cells derived from lung cells administered with only the solvent of the test substance; The method according to any one of claims 1 to 4.

6. The method according to any one of claims 1 to 5, wherein the expression level of mRNA of each gene of the discriminant indicator gene group in a cell derived from a lung cell administered with the test substance, a positive control substance, and / or a negative control substance is quantified using a pair of primers, a probe, or a single-stranded circular template comprising or consisting of a nucleotide sequence of SEQ ID NO: 1 and / or 2, 3 and / or 4, 5 and / or 6, 7 and / or 8, 9 and / or 10, 11 and / or 12, 13 and / or 14, 15 and / or 16, 17 and / or 18, 19 and / or 20, 21 and / or 22, 23 and / or 24, 25 and / or 26, 27 and / or 28, 29 and / or 30, 31 and / or 32, 33 and / or 34, and 35 and / or 36, or a nucleotide sequence complementary to said nucleotide sequence.

7. The method according to claim 6, wherein the expression level of mRNA of each gene of the group of discriminant indicator genes in cells derived from lung cells administered the test substance, positive control substance and / or negative control substance is quantified by PCR using primer pairs of SEQ ID NOs: 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, and 35 and 36.

8. The method according to any one of claims 1 to 7, wherein the cell derived from a lung cell is an alveolar macrophage.

9. The method of claim 8, wherein the alveolar macrophages are mouse MH-S cells.

10. The method according to any one of claims 5 to 9, wherein the internal standard gene is selected from the group consisting of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin, hypoxanthine phosphoribosyltransferase 1 (HPRT1), β2 microglobulin (B2M), and hydroxymethylbilane synthase (HMBS).

11. 11. A primer pair, probe or single-stranded circular template comprising or consisting of the nucleotide sequence of SEQ ID NO: 1 and / or 2, 3 and / or 4, 5 and / or 6, 7 and / or 8, 9 and / or 10, 11 and / or 12, 13 and / or 14, 15 and / or 16, 17 and / or 18, 19 and / or 20, 21 and / or 22, 23 and / or 24, 25 and / or 26, 27 and / or 28, 29 and / or 30, 31 and / or 32, 33 and / or 34, and 35 and / or 36, or a nucleotide sequence complementary thereto, for use in the method of any one of claims 1 to 10.

12. 11. A kit comprising a pair of primers, a probe or a single-stranded circular template comprising or consisting of the nucleotide sequence of SEQ ID NO: 1 and / or 2, 3 and / or 4, 5 and / or 6, 7 and / or 8, 9 and / or 10, 11 and / or 12, 13 and / or 14, 15 and / or 16, 17 and / or 18, 19 and / or 20, 21 and / or 22, 23 and / or 24, 25 and / or 26, 27 and / or 28, 29 and / or 30, 31 and / or 32, 33 and / or 34, and 35 and / or 36, or a nucleotide sequence complementary to said nucleotide sequence, for use in the method of any one of claims 1 to 10.

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