JP2013031371A - Method of evaluating biological effect of chemical substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve such a problem that it is difficult to evaluate hepatotoxicity detected by a hematological or pathological method with limited markers.SOLUTION: The change of an external environment causes acute alteration of in vivo gene expression, and identification of a gene set for determining a toxicity of a living body leads to quick and precise detection of the toxicity of the living body before occurrence of the toxicity or before positive proof of the toxicity by a pathological examination. Thus, a method for detecting or predicting the toxicity of the living body using the new gene set, a kit for the method, a method for treating the toxicity, and a method for confirming a drug candidate of the toxicity of the living body, are provided.

Description

  The present invention relates to a method for detecting, diagnosing, predicting and / or treating an effect of a chemical substance on a living body, toxicity, and a kit for detecting or predicting biotoxicity. In particular, the present invention relates to a method for detecting and predicting the toxicity of a chemical substance using the influence of the chemical substance on the living body as an index, a gene expression analysis means for helping to confirm the effectiveness of the treatment for hepatotoxicity, and a result thereof. Regarding use.

  A huge number of chemical substances are used in the environment where human beings live, and new chemical substances are still being developed year by year. However, when these chemical substances are released into the environment, there is a problem of having a harmful effect on ecosystems including the human body. In particular, the environmental pollution caused by chemical substances affects the human body. It has become a problem. In order to prevent accidents caused by harmful effects of new chemical substances on the human body and to ensure safety, information such as the presence / absence, strength, and target organs of these chemical substances should be investigated and understood in advance. It is important to keep From such a perspective, ministries and agencies that approve, approve, and register new chemical substances are required to conduct certain toxicity tests when reporting new chemical substances. Regulations have been made.

  Previous chemical substance risk assessment is a simple and simple test using bacteria, which is a test method introduced in Japan's “Chemical Substances Examination Regulation Law”, etc. based on internationally standardized test methods such as OECD. And the knowledge acquired and accumulated through long-term toxicity tests using laboratory animals such as rats (see Non-Patent Document 1).

  In recent years, a genomic approach that shows rapid development has been pharmacogenomics (see Non-Patent Documents 2 and 3), which investigates the correlation between drug sensitivity and side effects using biomarkers for personalized medicine. As with Nutrigenomics (Non-patent Document 4), which examines the effects of food on the living body by comprehensively analyzing fluctuations in the expression levels of mRNA and protein that occur as a result of ingestion, In particular, a technique called toxicogenomics, which has begun to be applied to the evaluation of its harmfulness, has been used (see Non-Patent Documents 5 to 7).

  These genomic methods have made it possible to evaluate biological phenomena from an enormous and diverse perspective that cannot be obtained by conventional methods by using all genes as individual parameters.

  As a method for evaluating the toxicity of chemical substances using gene expression variation analysis, a method for detecting toxic substances using yeast (see Patent Documents 1 and 2), a method for determining genotoxicity using cells (see Patent Document 3), Method for detecting developmental neurotoxicity using mammals (see Patent Documents 4 to 6), method for predicting carcinogens using mammals (see Patent Documents 7 and 8), method for predicting developmental toxicity using mammals ( (See Patent Document 9).

Japanese Patent No. 4022610 Japanese Patent No. 4475373 Japanese Patent No. 4573876 JP 2006-115748 A JP 2009-232842 JP 2009-77701 A JP 2009-159852 A JP 2007-54022 A JP 2010-111833 A

Non-clinical trial manual (ELC Corporation) (2001) Alison H. Harrill et al., Expert Opin. Drug Metab. Tosicol. November; 4 (11): 1379-1389 (2008) Elisa Giovannetti et al., Mol. Cancer Ther. 5 (6): 1387-1394 (2006). Licia Iacoviello et al., Genes Nutr. 3: 19-24 (2008) Preeti Chavan et al., Evid Based Complement Alternat Med. Dec; 3 (4): 447-457 (2006) Watanabe YAKUGAKU ZASSHI: 127 (12): 1967-1974 (2007) Uehara, Takeki et al., Mol. Nutr. Food Res. 54: 218-227 (2010)

  Conventional risk assessment of chemical substances was based on knowledge acquired and accumulated through simple and simple tests using bacteria and long-term toxicity tests using laboratory animals such as rats. However, the biological information obtained by these conventional toxicological methods is limited in the types of knowledge, and long-term toxicity studies have problems in terms of cost and efficiency. The establishment of a new method that can solve this problem has been desired.

  In particular, conventional 28-day repeated dose toxicity tests using laboratory animals such as rats mainly consist of hematological examinations and histopathological examinations, and their biological information is limited. In addition, the evaluation by histopathological examination is likely to depend on the subjectivity of the person who made the decision, and to evaluate the toxicity between different chemical substances, such as using different expressions despite seeing the same pathology. There were few objective indicators.

  An object of the present invention is to provide one objective index for simply and reliably detecting the hepatotoxicity of a chemical substance.

  The present inventor identified a gene whose expression was significantly altered in the liver after repeated administration of 3,4-xylidine (CAS registry number 95-64-7) to rats for 28 days, compared to the control group, The present invention has been completed.

That is, the present invention provides the following.
1. A method for evaluating the toxicity of a test chemical substance by measuring a gene expression level after exposing the test chemical substance to a living body or cells for a predetermined period,
(A) Prepare a plurality of cell samples derived from experimental animals or livers, and use a part of the test sample as the test sample after leaving the test chemical substance exposed to the test chemical substance for a predetermined period. Using as a reference sample a liver or liver-derived cell sample after treatment or exposure to said chemical solvent;
(B) About the said test | inspection sample, the expression level of the gene with respect to the arbitrary 1 or more selection biological response gene group selected from the biological response gene group as a gene group which has a base sequence shown by sequence number 1-74 is shown. A first gene expression level measurement step to measure,
(C) a second gene expression level measurement step for measuring the expression level of the gene for the selected biological response gene group for the reference sample;
(D) The gene expression levels measured in the first gene expression level measurement step and the second gene expression level measurement step are compared for each corresponding gene, and the test chemistry is based on the difference in the expression level of the gene. Evaluating the toxicity of the substance;
A method for evaluating the toxicity of a chemical substance, comprising:
2. 2. The toxicity evaluation method for a chemical substance according to the above 1, wherein the biological response gene group is a gene group having a base sequence represented by SEQ ID NOs: 1 to 59.
3. 2. The chemical substance toxicity evaluation method according to 1 above, wherein the biological response gene group is a gene group having a base sequence represented by SEQ ID NOs: 1 to 19.
4). 2. The chemical substance toxicity evaluation method according to 1 above, wherein the biological response gene group is a gene group having a base sequence represented by SEQ ID NOs: 1 to 13.
5). The expression level of the gene is measured using an expression level in a reporter gene including a sequence encoding a reporter protein linked to a promoter sequence in each biological response gene in the biological response gene group as an index, The toxicity evaluation method for a chemical substance according to any one of 1 to 4 above.
6). 6. A nucleic acid construct comprising a reporter gene used in the method according to 5 above, a vector comprising the same, or a transformed cell in which these are introduced into a host cell, the reporter linked to the promoter sequence of the biological response gene A nucleic acid construct comprising a sequence encoding a protein, a vector containing the nucleic acid construct, or a transformed cell in which these are introduced into a host cell.
7). 7. The transformed cell according to 6 above, wherein the host cell is an animal cell, a stem cell, or an embryonic stem cell.
8). A method for determining and predicting the toxicity of a test chemical substance by detecting the influence of the chemical substance on the living body at the gene expression level,
(A) administering (exposing) a predetermined amount of a chemical substance known to have hepatotoxicity to a biological or liver-derived cell sample for a predetermined period;
(B) administering (exposing) a predetermined amount of a chemical substance known not to have liver toxicity to a cell sample derived from a living body or a liver for a predetermined period;
(C) administering (exposing) a predetermined amount to a cell sample derived from a living body or a liver for a predetermined period using the chemical solvent as a control;
(D) An mRNA is isolated from the liver of the living body or a cell sample derived from the liver, and any one or more selected from the group of biological response genes as a group of genes having the base sequences of SEQ ID NOs: 1 to 74 A measurement step for measuring a gene expression level with respect to a biological response gene;
(E) collecting the gene expression level together with the corresponding chemical substance, exposure amount, and exposure period as gene expression data;
(F) exposing a test chemical substance to a living or liver-derived cell sample at an appropriate concentration for a certain period of time;
(G) isolating mRNA from the living body-derived liver or the liver-derived cell sample, and measuring a gene expression level for the biological response gene selected in the step (D);
(H) collecting the gene expression level obtained in (G) as gene expression data together with the test chemical substance, the exposure amount and the exposure period;
(I) comparing the gene expression data collected in (H) with the corresponding gene expression data for verification collected in (E);
A method for evaluating the toxicity of a chemical substance, comprising:
9. 9. The chemical substance toxicity evaluation method according to 8, wherein the biological response gene group is a gene group having a base sequence represented by SEQ ID NOs: 1 to 59.
10. 9. The chemical substance toxicity evaluation method according to 8, wherein the biological response gene group is a gene group having a base sequence represented by SEQ ID NOs: 1 to 19.
11. 9. The chemical substance toxicity evaluation method according to 8, wherein the biological response gene group is a gene group having a base sequence represented by SEQ ID NOs: 1 to 13.
12 Of the above 8 to 11, wherein the comparison of the gene expression data is a difference in gene expression level between a test chemical exposure group and a chemical exposure group known not to have liver toxicity The toxicity evaluation method of the chemical substance as described in any one.
13. The comparison of the gene expression data is a cluster analysis using as an index the expression profile of the biological response gene group in a chemical substance exposure group and a chemical substance exposure group that is known not to have liver toxicity. The toxicity evaluation method for a chemical substance according to any one of 8 to 11 above.
14 Comparison of the gene expression data is characterized by using a correlation coefficient of an expression profile of the biological response gene group in a chemical substance exposure group and a chemical substance exposure group known to have no hepatotoxicity as an index. The toxicity evaluation method for a chemical substance according to any one of 8 to 11 above.
15. Chemicals known to have liver toxicity include 2-butanone oxime (CAS registry number 96-29-7), 3-cyanopyridine (CAS registry number 100-54-9), sulfolane (CAS registry number 126-33). -0), 2-isopropoxyethanol (CAS registration number 109-59-1), hydrazine monohydrate (CAS registration number 7803-57-8), 4-ethylmorpholine (CAS registration number 100-74-3) , 3-methoxy-3-methyl-1-butanol (CAS registration number 55539-66-3), o-dichlorobenzene (CAS registration number 95-50-1), 3,4-xylidine (CAS registration number 95-64) -7), N-methylaniline (CAS registration number 100-61-8), tolylene diisocyanate (CAS registration number 26471-62-5), 2- (dibutylamino) ethanol (CAS registration number 102-81-8) ), P-cumylphenol (CAS registration number 599-64-4), m-cresol (CAS registration number 108-39-4), 2,3-dimethylaniline (CAS registration number 87-59-2), N , N'-Dicyclohexylcarbodi Mido (CAS registration number 538-75-0), diheptyl phthalate (CAS registration number 3648-21-3), tetrabromoethane (CAS registration number 79-27-6), p-ethylphenol (CAS registration number 123- 07-9), 2,4-di-tert-butylphenol (CAS registration number 96-76-4), 3,5-xylidine (CAS registration number 108-69-0), 1,3-dibromopropane (CAS registration) 109-64-8), 1-bromo-3-chloropropane (CAS registration number 109-70-6), pseudocumene (CAS registration number 95-63-6), 1,4-dibromobenzene (CAS registration number 106- 37. The method for evaluating toxicity of a chemical substance according to any one of 8 to 11, wherein the chemical substance is any one or more chemical substances selected from 37-6).
16. Chemicals known not to have liver toxicity include 2- (2-aminoethylamino) ethanol (CAS registration number 111-41-1), tetrahydrofurfuryl alcohol (CAS registration number 97-99-4), Methacrylamide (CAS registration number 79-39-0), ethyl trimethylammonium methacrylate (CAS registration number 5039-78-1), benzyltrimethylammonium chloride (CAS registration number 56-93-9), m-nitrobenzenesulfonic acid Sodium (CAS registration number 127-68-4), sodium 1-naphthylamine-4-sulfonate tetrahydrate (CAS registration number 130-13-2), dibutyl adipate (CAS registration number 105-99-7), N, N-dimethylbenzylamine (CAS registration number 103-83-3), n-hexadecane (CAS registration number 544-76-3), dicyclohexylamine (CAS registration number 101-83-7), and 2-amino- Select from 5-methylbenzenesulfonic acid (CAS registration number 88-44-8) 12. The chemical substance toxicity evaluation method according to any one of 8 to 11, wherein the chemical substance toxicity evaluation method is any one or more chemical substances.
17. The gene expression level is measured by one of RT-PCR, Real Time PCR, iAFLP (Introduced Amplified Fragment Length Polymorphism), LAMP (Loop-Mediated Isothermal Amplification), nCounter Analysis system, and hybridization. 17. The chemical substance toxicity evaluation method according to any one of 1 to 16, wherein a method is used.
18. 18. The chemical substance toxicity evaluation method according to 17 above, wherein the hybridization method is a microarray method or a blot method.
19. 19. The method for evaluating toxicity of a chemical substance according to the above 18, wherein the probe used in the microarray method or blotting method is a nucleotide or a protein.
20. 20. The method for evaluating toxicity of a chemical substance according to the above 19, wherein the nucleotide is mRNA, cDNA, or synthetic oligonucleotide.
21. 21. The method for evaluating toxicity of a chemical substance according to 19 or 20, wherein the nucleotide is a labeled nucleotide.
22. The measurement of the gene expression level is performed by measuring the presence or amount of a nucleic acid corresponding to the biological response gene or a protein encoded by the biological response gene. The toxicity evaluation method of the chemical substance as described in any one of them.
23. The method for evaluating toxicity of a chemical substance according to the item 22, wherein the protein is measured by an immunological method.
24. 24. The toxicity of a chemical substance according to 23, wherein the immunological method is based on a method for detecting an immunological complex of a specific antibody against a protein encoded by the biological response gene or a fragment thereof and a target protein. Evaluation method.
25. 25. The chemical substance toxicity evaluation method as described in 24 above, wherein the specific antibody is selected from a monoclonal antibody, a polyclonal antibody, a chimeric antibody, and an antibody fragment.
26. 26. A toxicity determination kit for a chemical substance comprising a probe used in the method according to any one of 1 to 25, wherein the probe specifically hybridizes to the biological response gene or a transcription product thereof. A chemical toxicity evaluation kit comprising a molecule having a sequence.
27. 27. The chemical substance toxicity evaluation kit according to 26, wherein the probe is a nucleotide or a protein.
28. 28. The chemical substance toxicity evaluation kit according to 27, wherein the nucleotide is mRNA, cDNA, or a synthetic oligonucleotide.
29. 29. The chemical substance toxicity evaluation kit according to 28, wherein the nucleotide hybridizes with a sense strand or an antisense strand of the biological response gene and has 10 to 100 bases.
30. 30. The chemical substance toxicity evaluation kit according to 28 or 29, wherein the nucleotide is a labeled nucleotide.
31. 31. The chemical substance toxicity evaluation kit according to 30 above, wherein the probe is a protein that is an antibody and / or an aptamer.
32. 32. The chemical substance according to any one of 26 to 31, wherein the probe includes a DNA microarray, a DNA chip, a protein chip, or an antibody chip in which any one or more are fixed to a solid support. Toxicity evaluation kit.
33. 33. The chemical substance toxicity evaluation kit according to 32, wherein the solid support is glass, silicon, plastic, or a biological membrane.

  According to the present invention, whether a chemical substance is toxic to a living body by comparing gene expression patterns in a liver sample after administration of the chemical substance to a living body or a liver-derived cell sample after exposure to the chemical substance. Whether or not can be easily determined or predicted.

It is a figure which shows a hierarchical cluster analysis result based on the expression variation pattern of the gene which has a base sequence shown by sequence number 1-74. It is a figure which shows a hierarchical cluster analysis result based on the expression variation pattern of the gene which has a base sequence shown to sequence number 1-59. It is a figure which shows a hierarchical cluster analysis result based on the expression variation pattern of the gene which has a base sequence shown by sequence number 1-19. It is a figure which shows a hierarchical cluster analysis result based on the expression variation pattern of the gene which has a base sequence shown by sequence number 1-13.

  Unless otherwise specified, the terms used in this application, including the specification and claims, are generally understood by those having ordinary skill in the art to which this invention belongs (those skilled in the art). Has the same meaning.

  Those skilled in the art will recognize many methods and materials equivalent or similar to those described herein. However, the present invention is not limited to the methods and materials described herein.

  It is desirable that the dose of the test chemical is an amount that moderately increases or decreases the gene expression level in the test animal or cells exposed to the test chemical. For example, a maximum dose less than the lethal dose of the test animal or cell is desirable, and it can be determined based on the LD50 value of the test chemical substance for the test animal.

  Mammals such as rats, mice, hamsters, guinea pigs, rabbits, dogs, monkeys, etc. can be used as test animals to which test chemical substances (test groups) or their solvents (control groups) are administered. . Moreover, cells derived from mammals such as rats, mice, hamsters, guinea pigs, rabbits, dogs, monkeys, and humans can be used as the cells to be targeted.

  The administration period of the test chemical substance is desirably 1 to 90 days, more preferably 1 to 60 days, and even more preferably 1 to 28 days. However, 1 to 14 days may be used from the viewpoint of conducting a test more quickly. Absent. Administration is preferably several times a day, more preferably once a day.

  The administration method of the test chemical substance is not particularly limited. For example, general methods such as oral administration, intraperitoneal administration, and intravenous injection can be used.

  “Measurement of gene expression level” is not particularly limited as long as the expression level of the gene is detected or quantified. For example, mRNA or cDNA of the gene may be detected or quantified. Furthermore, the protein encoded by the gene may be detected or quantified. For detection or quantification of these, it is desirable to use a molecule that specifically binds to the gene or its gene product, peptide or protein. Although it does not restrict | limit especially with the molecule | numerator specifically couple | bonded with a gene or its gene product peptide or protein, It bind | bond | couples specifically with the nucleotide, DNA, cDNA, RNA, peptide, or protein which bind | bond | couples specifically with this gene. An antibody etc. can be illustrated. In addition, for detection or quantification of the expression level of the gene, mRNA or protein fragments or homologues of the gene may be used.

  The nucleotide sequences shown in SEQ ID NOs: 1 to 74 specify the gene by homology search using BLAST (URL; http://blast.ncbi.nlm.nih.gov/) of National Center for Biotechnology Information, for example. Is possible.

  "DNA microarray" refers to oligonucleotides and single-stranded or double-stranded DNA arranged on a glass substrate at high density, and "DNA microarray method" refers to fluorescent labeling on the DNA microarray. A technique for qualitatively and quantitatively measuring the type and amount of nucleic acid bound to DNA by hybridization with cDNA molecules.

  “Oligonucleotide” is a general term for molecules in which several nucleotides are polymerized.

  An “homologue” of an mRNA relates to a nucleotide that is substantially similar to the mRNA. “Substantially similar” is well understood by those skilled in the art and specifically has a sequence similarity of at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95%. It means having.

  A protein “homolog” is related to a peptide substantially similar to the mRNA. “Substantially similar” is well understood by those skilled in the art and specifically has a sequence similarity of at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95%. It means having.

  “Organ tissue or cell sample exposed to a chemical substance” means that the tissue or cell sample or the animal from which the sample was derived has been treated with a chemical substance.

  “Stem cells” refer to undifferentiated cells that have both the ability to self-replicate and the ability to create differentiated cells. Embryonic stem cells (ES cells), tissue stem cells, induced pluripotent stem cells (iPS cells) : Induced pluripotent stem cell), but is not limited thereto.

  “Promoter” refers to a DNA region involved in the efficiency of the transcription initiation reaction.

  A “reporter gene” is a gene that is substituted for measuring the function of a target factor, and is preferably a gene that can easily quantify the activity of a product. The reporter gene of the present invention includes a promoter sequence of a biological response gene and a sequence encoding a reporter protein operably connected to the promoter sequence. Examples of the reporter protein include chloramphenicol acetyltransferase (CAT), Firefly luciferase, Renilla luciferase, β-galactosidase, green fluorescent protein (GFP), blue fluorescent protein (CFP), yellow fluorescent protein (YFP) or red fluorescent protein (dsRed), but are not limited to these Absent.

  In the present invention, “linked to the promoter sequence of the biological response gene” means that the expression of the target gene is placed under the control of the promoter sequence, and usually the promoter sequence immediately upstream of the target gene. Are not necessarily adjacent to each other.

  A “vector” refers to a DNA that can incorporate foreign DNA and increase in a host cell in recombinant DNA technology. Examples include plasmids, phages, viruses, and yeast artificial chromosomes. It is not something that can be done.

  A “transformed cell” is also referred to as a transformant or a transformant, and is a characteristic of a donor cell generated by introducing DNA of a cell (donor cell) exhibiting a certain trait into a cell (donor cell) that does not exhibit it. Refers to cells that exhibit Examples of donor cells or recipient cells include prokaryotic cells, yeast, animal cells, plant cells, and insect cells.

  “Toxic effect” refers to an adverse effect on a living body, organ system, each organ, tissue, cell, or intracellular unit due to the presence of a chemical substance. Toxic effects can be physiological or physical symptoms, or perturbations such as cell or organ necrosis.

  “Sample” is preferably intended to include material derived from liver tissue and any bodily fluid such as blood, plasma, serum, lymph, ascites, urine, stool. However, the present invention is not limited to this.

  As used in this application, including the specification and claims, an “individual” shall mean a human individual, an animal, or a population or pool of individuals.

  “CAS registration number” means a numerical identification number unique to a chemical substance assigned by a chemical substance registration system operated and managed by CAS (Chemical Abstracts Service), a division of the American Chemical Society.

  The term “biological response gene” used in the claims and specification of the present application means a gene whose expression level fluctuates in vivo due to an external stimulus such as exposure to a chemical substance. “Group” means a combination of a plurality of biological response genes.

  Specific methods for detecting, measuring or quantifying the expression level of a gene include Northern blotting and dot blotting using labeled nucleotides, labeled cDNA or labeled RNA for probes that specifically bind to the gene. IAFLP (introduced Amplified Fragment Length Polymorphism) method, LAMP (Loop-Mediated Isothermal Amplification) method, PCR method, method of directly measuring mRNA molecules, and the like can be used. Examples of the PCR method include RT-PCR method, Real Time PCR method, and competitive PCR method.

  As the Real Time PCR method, for example, cDNA is synthesized from the total RNA or mRNA in a sample using reverse transcriptase, the target region is amplified by the PCR method using the cDNA as a template, and the production process of the amplification product is performed. A method for monitoring the real time in real time. Examples of the reagent to be monitored in real time include SYBR (registered trademark: Moleclar Probes) Green I, TaqMan (registered trademark: Applied Biosystems) probe, and the like.

  Examples of the competitive PCR method include, for example, a method of synthesizing cDNA from total RNA or mRNA in a sample using a reverse transcriptase and reacting the cDNA and an internal standard DNA in the same reaction tube. Examples include a method of reacting by adding an RNA internal standard together with mRNA during the reaction. In addition, the sequence of the internal standard gene may be, for example, a sequence homologous to the sequence of the gene to be amplified or a non-homologous sequence.

  Furthermore, specific methods for detecting or quantifying gene expression levels include methods using DNA microarrays, DNA chips, antibody arrays, and the like. A DNA microarray or DNA chip having one or more nucleotides or cDNAs of the gene immobilized thereon is used.

  The nucleotide or cDNA may be a part corresponding to a part of the gene.

  The labeling reagent used for labeling the probe is, for example, radioisotopes [125I], [131I], [3H], [14C], [32P], [35S], an enzyme β-galactosidase, β -Glucosidase, alkaline phosphatase, peroxidase, and cyanine fluorescent dye fluorescent dyes that are fluorescent substances (for example, Cy2, Cy3, Cy5, Cy5.5, Cy7, Cyanine3, Cyanine5, etc.) can be used.

  In addition, examples of the Real Time PCR method include a method of monitoring PCR amplification products in real time using cDNA synthesized by reverse transcriptase from total RNA or mRNA in tissues or cells as a template. As a monitoring reagent for real-time PCR, for example, SYBR Green I or TaqMan probe is used.

  Usually, a DNA microarray or DNA chip is an array or chip in which probes are fixed on a support, and the support of the DNA microarray or DNA chip may be any one that can be used for hybridization. A substrate such as glass, silicon, or plastic, a nitrocellulose film, a nylon film, or the like can be used.

  The DNA microarray refers to one in which at least one cDNA fragment or oligo DNA complementary to the full length of a gene included in a biological response gene group or a partial sequence thereof is fixed to a fixed support. The complementary oligo DNA here is generally 25 to 100 bases in length, but is not necessarily limited thereto.

  There are no particular restrictions on the method of using the DNA microarray or DNA chip. For example, when mRNA is purified from a biological sample and a reverse transcription reaction is performed using the mRNA as a template, a labeled cDNA can be obtained by using an appropriately labeled primer or labeled nucleotide. Hybridization is performed between the labeled cDNA and the probe of the present invention immobilized on the surface of a DNA microarray or DNA chip, and the results of hybridization with a test sample and hybridization with a control sample are compared. By detecting the presence or absence of the gene or measuring the expression level, organ toxicity can be detected or predicted.

  The polypeptide or protein corresponding to the gene is an expression product of the biological response gene, and the sequence information of the amino acid sequence of the polypeptide or protein can be approached by each accession number in the NCBI gene database.

  The method for detecting or quantifying the polypeptide or protein is not particularly limited as long as it is a method for detecting or quantifying a predetermined polypeptide or protein. For example, an antibody or aptamer that specifically binds to the polypeptide or protein can be used. As the antibody, a monoclonal antibody, a polyclonal antibody, a single chain antibody, a humanized antibody, a chimeric antibody, two epitopes can be used simultaneously. Examples include bifunctional antibodies that can be recognized. These antibodies can be produced using the polypeptide or protein or a fragment thereof as an antigen using conventional protocols. Aptamers are nucleic acid molecules that specifically bind to molecules such as proteins and amino acids.

  When detecting or quantifying the polypeptide or protein present in a test sample using an antibody that specifically binds to the polypeptide or protein, immunoprecipitation, electrochemiluminescence, RIA (Radioimmunoassay), Known immunological methods such as an ELISA (Enzyme-liked immunosorbent assay) method and a fluorescent antibody method can be used.

  As a criterion for the determination, the expression level of the gene (or the expression level of the polypeptide or protein corresponding to the gene) present in the test sample is present in the normal control sample (or The expression level is higher or lower than the expression level of the polypeptide or protein corresponding to the gene. For example, when a t-test is performed on the results of measuring the expression level of three or more samples per group, P <0.05, more preferably P <0.01, still more preferably P <0.001, and even more preferably P < An example is 0.0001.

  The test method is not limited to the t-test, and may be a U-test, F-test, Mann-Whitney test or Wilcoxan signed rank test. Moreover, it is not limited to a test | inspection, For example, you may use the difference of the average value of the expression level of each group.

  The reference value does not need to be measured each time the expression level in the test sample is measured. For example, the expression level of a gene present in a normal control sample in various biological samples is measured in advance. Comparison can be made using measured values.

  Changes in gene expression levels include not only direct reactions between specific chemicals and living tissues, but also secondary reactions as a result of organ damage.

  A gene included in the biological response gene group can be used as a marker in any mammal such as human, rat, mouse, rabbit, or monkey. Preferably, genes included in the biological response gene group are used as markers in rats or mice.

  The type of animal is not particularly limited. For example, in the case of a rat, it may be a Sprague Dawley rat, a Wistar rat, etc., and may be male or female.

  Hereinafter, the chemical substance toxicity determination / prediction method, nucleic acid composition, vector, transformed cell, method for creating a reference gene expression database, and chemical substance toxicity determination kit according to the present invention will be described more specifically by way of examples. However, the technical scope of the present invention is not limited to these examples.

  The biological response gene group used in the method for detecting or predicting the toxic effect of the present invention includes 3,4-xylidine (CAS registration number 95-64-7) and male Sprague Dawley rat (6 weeks old) (Japan). This is a group of genes whose expression levels are significantly changed in the liver by repeated administration to Charles River) for 28 days.

  The biological response gene group used in the present invention is obtained by the following method. Here, the “expression level” need not be an absolute amount, but may be a relative amount.

<Gene expression database>
Creation of a gene expression database according to the present invention
(1) For various chemical substances, determine appropriate doses that will not cause rats to die,
(2) Repeated exposure of a chemical substance of appropriate concentration to a rat etc. for a certain period,
(3) Remove each organ from the exposed living body,
(4) mRNA is isolated from the removed organ,
(5) Measure the expression level of a specific gene by DNA microarray method,
(6) The obtained gene expression level is compiled as a gene expression database together with chemical substances, their concentrations, and exposure times, and the above six steps are performed.

<Animal test>
Five-week-old Crl: CD (SD) rats (male) were prepared, placed in polycarbonate cages, and raised in an air-conditioned animal rack (trade name). The air conditioning animal rack was set to a temperature of 22 ° C. and a humidity of 55%, and the lighting was set to a 12-hour cycle of light period 7:00 to 19:00 and dark period 19:00 to 7:00. Water was constantly fed tap water through a water purifier using a water supply bottle, and feed was constantly fed solid feed. A one-week habituation quarantine period was established before the start of the experiment.

  2-Butanone oxime, 37 kinds of chemical substances registered in the “National Database on Toxicology of Chemicals” (http://dra4.nihs.go.jp/mhlw_data/jsp/SearchPage.jsp) , 3-cyanopyridine, 2- (2-aminoethylamino) ethanol, tetrahydrofurfuryl alcohol, methacrylamide, sulfolane, 2-isopropoxyethanol, hydrazine monohydrate, 4-ethylmorpholine, ethyl trimethylammonium chloride , Benzyltrimethylammonium chloride, sodium m-nitrobenzenesulfonate, sodium 1-naphthylamine-4-sulfonate tetrahydrate, 3-methoxy-3-methyl-1-butanol, o-dichlorobenzene, 3,4-xylidine, N-methylaniline, tolylene diisocyanate, 2- (dibutylamino) ethanol, p-cumylphenol, m -Cresol, 2,3-dimethylaniline, N, N'-dicyclohexylcarbodiimide, diheptyl phthalate, tetrabromoethane, dibutyl adipate, p-ethylphenol, 2,4-di-tert-butylphenol, 3,5-xylidine , N, N-dimethylbenzylamine, 1,3-dibromopropane, n-hexadecane, 1-bromo-3-chloropropane, pseudocumene, dicyclohexylamine, 1,4-dibromobenzene, or 2-amino-5-methylbenzenesulfone The acid was orally administered to Sprague Dawley rats (6 weeks old, male) repeatedly for 28 days. As a normal control group, olive oil, water for injection or sesame oil was orally administered to Sprague Dawley rats (6 weeks old, male) repeatedly for 28 days. Three rats were used per group. The animal test is not limited to 28 days, and may be several days, for example.

  For the chemical substance administration liquid, a necessary amount of the chemical substance was weighed, and a solution or uniform suspension was prepared using an appropriate solvent (water for injection, olive oil, sesame oil, etc.). Oral administration was performed using a syringe for 2.5 mL or 5.0 mL equipped with a flexible oral sonde (trade name), but is not limited thereto. The solvent is 2-butanone oxime, 3-cyanopyridine, 2- (2-aminoethylamino) ethanol, tetrahydrofurfuryl alcohol, methacrylamide, sulfolane, 2-isopropoxyethanol, hydrazine monohydrate, 4- Ethylmorpholine, ethyltrimethylammonium methacrylate chloride, benzyltrimethylammonium chloride, sodium m-nitrobenzenesulfonate, sodium 1-naphthylamine-4-sulfonate tetrahydrate, and 3-methoxy-3-methyl-1-butanol are injected Using water (Otsuka Pharmaceutical Co., Ltd.), o-dichlorobenzene, 3,4-xylidine, N-methylaniline, tolylene diisocyanate, 2- (dibutylamino) ethanol, p-cumylphenol, m-cresol 2,3-dimethylaniline, N, N'-dicyclohexylcarbodiimide, Diheptyl formate, tetrabromoethane, dibutyl adipate, p-ethylphenol, 2,4-di-tert-butylphenol, 3,5-xylidine, N, N-dimethylbenzylamine, 1,3-dibromopropane and n- Hexadecane uses olive oil (manufactured by Kosuge Pharmaceutical Co., Ltd.), and 1-bromo-3-chloropropane, pseudocumene, dicyclohexylamine, 1,4-dibromobenzene, and 2-amino-5-methylbenzenesulfonic acid are sesame oil (Kosuge Pharmaceutical Co., Ltd.) was used. In addition, the solvent used for an administration liquid is not limited to these.

  The dose of each chemical substance is 100 mg / kg / day for 2-butanone oxime, 180 mg / kg / day for 3-cyanopyridine, 1,000 mg / kg / day for 2- (2-aminoethylamino) ethanol, tetrahydro Furfuryl alcohol 600 mg / kg / day, methacrylamide 150 mg / kg / day, sulfolane 700 mg / kg / day, 2-isopropoxyethanol 500 mg / kg / day, hydrazine monohydrate 30 mg / kg / day 4-ethylmorpholine 500 mg / kg / day, ethyl trimethylammonium methacrylate 1,000 mg / kg / day, benzyltrimethylammonium chloride 120 mg / kg / day, sodium m-nitrobenzenesulfonate 1,000 mg / kg / day 1-naphthylamine-4-sulfonic acid sodium tetrahydrate 1,000 mg / kg / day, 3-methoxy-3-methyl-1-butanol 1,000 mg / kg / day, o-dichlorobenzene 500 mg / kg / day day, 3,4-xylidine 250 mg / kg / day, N-methylaniline 125 mg / kg / day, Range isocyanate 300mg / kg / day, 2- (dibutylamino) ethanol 250mg / kg / day, p-cumylphenol 700mg / kg / day, m-cresol 1,000mg / kg / day, 2,3 -200 mg / kg / day for dimethylaniline, 300 mg / kg / day for N, N'-dicyclohexylcarbodiimide, 1,000 mg / kg / day for diheptyl phthalate, 200 mg / kg / day for tetrabromoethane, 1,000 for dibutyl adipate mg / kg / day, 1,000 mg / kg / day for p-ethylphenol, 300 mg / kg / day for 2,4-di-tert-butylphenol, 200 mg / kg / day for 3,5-xylidine, N, N- Dimethylbenzylamine 200 mg / kg / day, 1,3-dibromopropane 250 mg / kg / day, n-hexadecane 1,000 mg / kg / day, 1-bromo-3-chloropropane 300 mg / kg / day, pseudocumene 1,000 mg / kg / day, dicyclohexylamine 70 mg / kg / day, 1,4-dibromobenzene 300 mg / kg / day, 2-amino-5-methylbenzenesulfonic acid 1,000 mg / kg / day Calculate the dosing solution volume from the measured weight of the rat as a, it was administered to rats.

  Organs were collected approximately 24 hours after the last dose of chemical. Specifically, the rats were exsanguinated (total blood collection) from the abdominal aorta under anesthesia and euthanized, and then the liver was collected and immediately frozen in liquid nitrogen. The frozen liver was pulverized by homogenization in an ISOGEN (Nippon Gene) solution.

<Extraction of total RNA>
Extraction of total RNA from liver tissue was performed according to the recommended protocol using ISOGEN reagent (Nippon Gene).

<Preparation of nucleic acid sample>
Sample mRNA was prepared from total RNA extracted from liver tissue using ISOGEN reagent (Nippon Gene) using a Poly (A) Pure kit (Ambion) according to the protocol recommended by each company.

<Production of microarray>
A microarray was prepared using a rat gene fragment library (manufactured by Microdiagnostic). The rat gene fragment library contained a synthetic oligonucleotide having a base sequence represented by SEQ ID NOs: 1 to 74. In addition, although there are no limitations on the microarray manufacturing method and conditions, for example, the manufacturing method described in (Schena, M. et.al., Science, 270, 467-470. (1995)) can be used.

  The rat gene fragment library was printed on a slide glass (manufactured by Matsunami Glass Industrial Co., Ltd., HA-coated slide glass) using an ultra-small volume dispensing apparatus (manufactured by Microdiagnostic) to prepare a microarray. The microarray was allowed to stand at 80 ° C. for 1 hour in a gas-phase incubator, and further irradiated with 120 mJ ultraviolet rays using a UV crosslinker (Hoefer, UVC500).

<Post-processing of microarray>
The post-treatment of the microarray was performed by the method described in the patent publication (Japanese Patent No. 4190899).

<Synthesis of labeled cDNA>
1.5 μg of the mRNA was labeled with a nucleic acid labeling / hybridizing reagent (manufactured by Microdiagnostic), reverse transcriptase SuperScriptII (registered trademark: Life Technologies) (manufactured by Invitrogen), cyanine5-deoxyuridinetriphosphate (Cyanine5-dUTP) (manufactured by Perkin Elmer) ) Was used to prepare labeled cDNA. On the other hand, a rat common reference (manufactured by Microdiagnostic) was used as a control. For common reference, nucleic acid labeling / hybridization reagent (Microdiagnostic), reverse transcriptase SuperScriptII (Invitrogen), Cyanine3-deoxyuridinetriphosphate (Cyanine3-dUTP) (Perkin Elmer) is used and labeled cDNA Was made. The production method followed the protocol recommended by each company.

<Preparation of labeled probe>
After mixing these labeled cDNAs, that is, a sample labeled with Cyanine5-dUTP and a control reference labeled with Cyanine3-dUTP in the same test tube, MicropureEZ (Millipore) and MicroconYM30 (registered trademark: Millipore) (Millipore) ). Finally, 15 μl was prepared using the hybridization buffer and pure water attached to the nucleic acid labeling / hybridization reagent.

<Hybridization>
The solution was heat denatured by heating at 99 ° C. for 5 minutes, then dropped onto a DNA microarray and stored in a hybridization cassette (manufactured by Microdiagnostic). The hybridization cassette was placed in a gas-phase incubator (manufactured by Sanyo Electric Biomedica Co., Ltd.) and kept warm at 42 ° C. for about 20 hours. By this operation, the labeled cDNA contained in the sample specifically binds to the complementary oligo DNA on the DNA microarray.

<Washing>
The slide glass was taken out from the hybridization cassette, and the slide glass was washed using a hybridization washing solution attached to a nucleic acid labeling / hybridization reagent (manufactured by Microdiagnostic) according to the protocol recommended by the company.

<Detection and digitization of fluorescence intensity>
The expression level of each gene can be estimated by measuring the fluorescence intensity of the labeled cDNA bound to the oligo DNA immobilized on the DNA microarray. Fluorescence is measured on the cleaned glass slide using the scanner GenePix4000B (Axon Instrument), optically evaluated using the analysis software GenePixPro (Axon Instrument) attached to the scanner, and the relative value of fluorescence intensity (Cyanine5 / Cyanine3) Quantified. That is, the fluorescence intensity of each oligo DNA spot immobilized on the DNA microarray was measured separately, and the fluorescence intensity was expressed as a relative ratio (log ₂ ratio) to the human common reference. In addition, the background was calculated from the fluorescence intensity at a place other than the spot, and was subtracted from the fluorescence intensity of each spot as noise. Furthermore, the analysis of calculating the fluorescence intensity of the sample / the fluorescence intensity of the common reference was performed. That is, since the gene expression level of each sample is detected as a relative ratio to the common reference, a plurality of samples can be simply compared side by side. The numerical values obtained in this way were accumulated to form a database.

<Calculation of secondary ratio>
Next, an average value of all the control groups was calculated, and a relative value (referred to as “secondary ratio”) with respect to the average value was calculated for each sample. The following calculations were all performed using the secondary ratio.

<Selection of chemicals that affect the liver>
37 types of chemical substances registered in the “Existing Chemical Substance Toxicity Database” (National Institute of Food and Drug Hygiene): 2-butanone oxime, 3-cyanopyridine, 2- (2-aminoethylamino) Ethanol, tetrahydrofurfuryl alcohol, methacrylamide, sulfolane, 2-isopropoxyethanol, hydrazine monohydrate, 4-ethylmorpholine, ethyl trimethylammonium methacrylate, benzyltrimethylammonium chloride, sodium m-nitrobenzenesulfonate, 1- Sodium naphthylamine-4-sulfonate tetrahydrate, 3-methoxy-3-methyl-1-butanol, o-dichlorobenzene, 3,4-xylidine, N-methylaniline, tolylene diisocyanate, 2- (dibutylamino ) Ethanol, p-cumylphenol, m-cresol, 2, 3-dimethylaniline, N, N'-dicyclohexylcarbodiimide, diheptyl phthalate, tetrabromoethane, dibutyl adipate, p-ethylphenol, 2,4-di-tert-butylphenol, 3,5-xylidine, N, N- Dimethylbenzylamine, 1,3-dibromopropane, n-hexadecane, 1-bromo-3-chloropropane, pseudocumene, dicyclohexylamine, 1,4-dibromobenzene, 2-amino-5-methylbenzenesulfonic acid were selected.

<Extraction of gene group>
Comparison of the livers of rats administered repeatedly with 3,4-xylidine for 28 days and the livers of control rats administered with the solvent, and the Student's t-test for the relative expression ratio of logarithmic transformation of each gene Was calculated. When a group of genes having an absolute value of an average difference in expression level between each chemical substance administration group and the control group of 1.0 or more and a P value of less than 0.01 was extracted, 74 probes were present (SEQ ID NO: 1). A gene group having a base sequence represented by ~ 74). Tables 1 to 14 show the expression information of the gene group corresponding to the 74 probe (the gene group having the base sequence shown in SEQ ID NOs: 1 to 74), and the numerical values are expressed as secondary ratios. In the table, the “SEQ ID NO” column indicates the SEQ ID NO of the identified gene. The abbreviations in the table are “C1” for “injection water administration group used in the first experiment”, “2bo” for “2-butanone oxime administration group”, and “3cp” for “3-cyanopyridine”. “Administration group”, “2ae” is “2- (2-aminoethylamino) ethanol administration group”, “thf” is “tetrahydrofurfuryl alcohol administration group”, “C2” is “second experiment” `` Water injection group used for injection '', `` mca '' for `` methacrylamide administration group '', `` suf '' for `` sulfolane administration group '', `` 2ip '' for `` 2-isopropoxyethanol administration group '', `` hmh '' Is the “hydrazine monohydrate administration group”, “4em” is the “4-ethylmorpholine administration group”, “C3” is the “water injection group used for the third experiment”, and “mta” “Ethyltrimethylammonium methacrylate administration group” and “bac” are “benzyltrimethylammonium chloride”. `` Monium administration group '', `` mns '' is `` m-nitrobenzenesulfone sodium sodium administration group '', `` nat '' is `` 1-naphthylamine-4-sulfonic acid sodium tetrahydrate administration group '', `` mmb '' is `` “3-methoxy-3-methyl-1-butanol administration group”, “C4” “injection water administration group used in the fourth experiment”, “dcb” “o-dichlorobenzene administration group”, "34x" is "3,4-xylidine administration group", "nma" is "N-methylaniline administration group", "tdn" is "tolylene diisocyanate administration group", "2de" is "2- (Dibutylamino) ethanol administration group ”,“ C5 ”is“ Olive oil administration group used in the fifth experiment ”,“ pcp ”is“ p-cumylphenol administration group ”,“ mcs ”is“ `` m-cresol administration group '', `` 23d '' is `` 2,3-dimethylaniline administration group '', `` dhc '' is `` N, N'-dicyclohexylcarbodihydrate '' “Mid administration group”, “dhp” is “diheptyl phthalate administration group”, “C6” is “oliv oil administration group used in the 6th experiment”, “tbe” is “tetrabromoethane administration group” “Dba” is the “dibutyl adipate administration group”, “pep” is the “p-ethylphenol administration group”, “C7” is “the olive oil administration group used in the seventh experiment”, “ `` 24b '' is the `` 2,4-di-tert-butylphenol administration group '', `` 35x '' is the `` 3,5-xylidine administration group '', `` nda '' is the `` N, N-dimethylbenzylamine administration group '', “13d” is “1,3-dibromopropane administration group”, “nhd” is “n-hexadecane administration group”, “C8” is “sesame oil”, “bcp” is “1-bromo-3-chloropropane” `` Administration group '', `` tmb '' is `` pseudocumene administration group '', `` dha '' is `` dicyclohexylamine administration group '', `` 14d '' is `` 1,4-dibromobenzene administration group '' The "ams" represents "2-amino-5-methylbenzenesulfonic acid administration group". The number accompanying the abbreviation represents the individual.

  The experiment was divided into 8 times. In the first experiment, four kinds of chemical substances, 2-butanone oxime, 3-cyanopyridine, 2- (2-aminoethylamino) ethanol, and tetrahydrofurfuryl alcohol, were used for each injection. In the second experiment, five chemical substances, methacrylamide, sulfolane, 2-isopropoxyethanol, hydrazine monohydrate, and 4-ethylmorpholine, were dissolved in water for injection and administered. In the third experiment, ethyl trimethylammonium methacrylate chloride, benzyltrimethylammonium chloride, sodium m-nitrobenzenesulfone, sodium 1-naphthylamine-4-sulfonate tetrahydrate, 3-methoxy-3-methyl-1- 5 chemicals of butanol were dissolved in water for injection and administered in the fourth experiment. Five chemical substances (robenzene, 3,4-xylidine, N-methylaniline, tolylene diisocyanate, 2- (dibutylamino) ethanol) were dissolved in water for injection and administered. In the fifth experiment, p- Five chemical substances, cumylphenol, m-cresol, 2,3-dimethylaniline, N, N'-dicyclohexylcarbodiimide, and diheptyl phthalate, were each dissolved in olive oil and administered in the sixth experiment. Three chemical substances, bromoethane, dibutyl adipate, and p-ethylphenol, were dissolved in olive oil and administered. In the seventh experiment, 2,4-di-tert-butylphenol and 3,5-xylidine were used. , N, N-dimethylbenzylamine, 1,3-dibromopropane and n-hexadecane were dissolved in olive oil and administered. In the 8th experiment, 1-bromo-3- Roropuropan, pseudocumene, dicyclohexylamine, 1,4-dibromobenzene, was administered by dissolving 2-amino-5-five of chemicals methylbenzenesulfonic acid in sesame oil, respectively.

  In addition, abnormalities in the pathological findings of the liver were reported when 21 of the 37 chemical substances were repeatedly administered to rats for 28 days. “3-cyanopyridine administration group”, “4-ethylmorpholine administration group”, “2- (dibutylamino) ethanol administration group”, “m-cresol administration group”, “tetrabromoethane administration group”, “2,4 -Di-tert-butylphenol administration group, 1,3-dibromopropane administration group, 1-bromo-3-chloropropane administration group, 1,4-dibromobenzene administration group, "O-dichlorobenzene administration group" and "diheptyl phthalate administration group" show hepatocyte hypertrophy, hepatocyte fattening (decreasing change) and centrilobular single cell necrosis, and "3,5-xylidine administration group" External hematopoiesis and pigmentation in the “3,4-xylidine-administered group” include single-cell necrosis in addition to extramedullary hematopoiesis and pigmentation in the “2,3-dimethylaniline-administered group” and “p-cumylphenol-administered group” Bile duct proliferation, "2-butanone oxime administration group", "2-isopropoxyeta `` Administration group '', `` N-methylaniline administration group '', extramedullary hematopoiesis and pigmentation, `` hydrazine monohydrate administration group '', `` tolylene diisocyanate administration group '', Hepatocyte necrosis was reported in the “N, N′-dicyclohexylcarbodiimide group”. In addition, there were no significant changes in pathological findings, but the chemicals reported to be toxic to the liver in other tests were the "sulfolane administration group" and the "3-methoxy-3-methyl-1-butanol administration group" , “P-ethylphenol administration group” and “pseudocumene administration group”.

<Cluster analysis>
As an analysis method of gene expression data acquired by a DNA microarray, for example, cluster analysis can be mentioned. Cluster analysis is a statistical technique for grouping genes with similar gene expression change patterns. By defining similarity between data (for example, Euclidean distance) and using the similarity, chemical substances having similar gene expression patterns, that is, chemical substances having similar influence on gene expression are grouped.

  Hierarchical cluster analysis was performed based on the expression variation pattern of the gene having the base sequence shown in SEQ ID NOs: 1 to 74. Hierarchical cluster analysis was performed using analysis software “Expression View Pro” (manufactured by Microdiagnostic). Hierarchical cluster analysis can also be performed using software such as “cluster” and “treeview”. As a result, chemical substances could be roughly classified into four clusters. FIG. 1 shows the result of cluster analysis. In the figure, “C1” is “injection water administration group used in the first experiment”, “2bo” is “2-butanone oxime administration group”, and “3cp” is “3-cyanopyridine administration group”. "2ae" was used in the "2- (2-aminoethylamino) ethanol administration group", "thf" was used in the "tetrahydrofurfuryl alcohol administration group", and "C2" was used in the second experiment. `` Water injection group '', `` mca '' is `` methacrylamide group '', `` suf '' is `` sulfolane group '', `` 2ip '' is `` 2-isopropoxyethanol group '', `` hmh '' is `` hm '' “Hydrazine monohydrate administration group”, “4em” “4-ethylmorpholine administration group”, “C3” “injection water administration group used in the third experiment”, “mta” “methacrylic” `` Ethyl trimethylammonium chloride administration group '', `` bac '' is `` benzyltrimethylammonium chloride '' `` Group administration group '', `` mns '' is `` m-nitrobenzenesulfone sodium sodium administration group '', `` nat '' is `` 1-naphthylamine-4-sulfonic acid sodium tetrahydrate administration group '', `` mmb '' is `` “3-methoxy-3-methyl-1-butanol administration group”, “C4” “injection water administration group used in the fourth experiment”, “dcb” “o-dichlorobenzene administration group”, "34x" is "3,4-xylidine administration group", "nma" is "N-methylaniline administration group", "tdn" is "tolylene diisocyanate administration group", "2de" is "2- (Dibutylamino) ethanol administration group ”,“ C5 ”is“ Olive oil administration group used in the fifth experiment ”,“ pcp ”is“ p-cumylphenol administration group ”,“ mcs ”is“ `` m-cresol administration group '', `` 23d '' is `` 2,3-dimethylaniline administration group '', `` dhc '' is `` N, N'-dicyclohexylcarbodiimide '' “Yoh group”, “dhp” is “diheptyl phthalate administration group”, “C6” is “Olive oil administration group used in the 6th experiment”, “tbe” is “tetrabromoethane administration group” , “Dba” is the “dibutyl adipate administration group”, “pep” is the “p-ethylphenol administration group”, “C7” is the “olive oil administration group used in the seventh experiment”, “24b `` 2,4-di-tert-butylphenol administration group '', `` 35x '' `` 3,5-xylidine administration group '', `` nda '' `` N, N-dimethylbenzylamine administration group '', `` `` 13d '' is `` 1,3-dibromopropane administration group '', `` nhd '' is `` n-hexadecane administration group '', `` C8 '' is `` sesame oil administration group '', `` bcp '' is `` 1-bromo-3- `` Chloropropane administration group '', `` tmb '' is `` pseudocumene administration group '', `` dha '' is `` dicyclohexylamine administration group '', `` 14d '' is `` 1,4-dibromobenzene administration group '' The "ams" represents "2-amino-5-methylbenzenesulfonic acid administration group". The black and white bars under the abbreviations representing the sample names indicate the presence or absence of liver toxicity, with black indicating a chemical substance having liver toxicity and white indicating a chemical substance having no liver toxicity. Furthermore, black circles and white circles represent samples to be compared when specifying genes having the nucleotide sequences shown in SEQ ID NOs: 1 to 74, black circles represent chemical substance administration samples, and white circles represent control samples. Yes. “A” to “D” represent major clusters.

  In the cluster “A”, 3,4-xylidine administration group (34x) is 3 out of 3 individuals, o-dichlorobenzene administration group (dcb) is 3 out of 3 individuals, 1-bromo-3-chloropropane administration group (Bcp) is 3 out of 3 individuals, 1,3-dibromopropane administration group (13d) is 3 out of 3 individuals, tetrabromoethane administration group (tbe) is 3 out of 3 individuals, 1,4-dibromobenzene administration The group (14d) included 3 out of 3 individuals, and the 3-cyanopyridine administration group (3cp) included 3 out of 3 individuals. In the cluster “B”, the hydrazine monohydrate administration group (hmh) included 2 out of 3 individuals, and the diheptyl phthalate administration group (dhp) included 3 out of 3 individuals. In the cluster of “C”, N-methylaniline administration group (nma) is 3 out of 3 individuals, 3,5-xylidine administration group (35x) is 3 out of 3 individuals, p-cumylphenol administration group (pcp ) Is 3 out of 3 individuals, sulfolane administration group (suf) is 3 out of 3 individuals, p-ethylphenol administration group (pep) is 2 out of 3 individuals, pseudocumene administration group (tmb) is 3 out of 3 individuals, The tolylene diisocyanate administration group (tdn) included 2 out of 3 individuals, and the 2,4-di-tert-butylphenol administration group (24b) included 3 out of 3 individuals. All the chemical substances contained in the clusters “A” to “C” were known to have hepatotoxicity. The “D” cluster included other samples including the control group. This result means that the hepatotoxicity of the chemical substance can be evaluated by the expression variation pattern of the gene having the base sequence shown in SEQ ID NOs: 1 to 74.

  Next, the rat t-test was performed for the logarithmic transformation relative expression ratio of each gene by comparing the liver of a rat administered with repeated administration of 3,4-xylidine for 28 days with the liver of a control group administered with the solvent. The P value was calculated, and a gene group having an absolute value of the difference in the average value of the expression levels of 1.0 or more and a P value of less than 0.005 was extracted. Gene group having a base sequence). Hierarchical cluster analysis was performed based on the expression variation pattern of the gene having the base sequence shown in SEQ ID NOs: 1 to 59. As a result, chemical substances could be roughly classified into 8 clusters. FIG. 2 shows the result of cluster analysis. The abbreviations in the figure are the same as those in FIG.

  In the cluster “A”, the tetrabromoethane administration group (tbe) was 3 out of 3 individuals, the 3-cyanopyridine administration group (3cp) was 3 out of 3 individuals, and the 1,3-dibromopropane administration group (13d) Is 3 out of 3 individuals, 1-bromo-3-chloropropane administration group (bcp) is 3 out of 3 individuals, o-dichlorobenzene administration group (dcb) is 3 out of 3 individuals, 1,4-dibromobenzene administration group (14d) included 3 out of 3 individuals, and 3,4-xylidine administration group (34x) included 3 out of 3 individuals. The “B” cluster contained 3 out of 3 individuals in the diheptyl phthalate administration group (dhp). The cluster of “C” included 3 out of 3 individuals in the 3,5-xylidine-administered group (35x) and 3 out of 3 individuals in the N-methylaniline-administered group (nma). In the cluster “D”, the p-cumylphenol administration group (pcp) is 3 out of 3 individuals, the sulfolane administration group (suf) is 3 out of 3 individuals, and the pseudocumene administration group (tmb) is 3 out of 3 individuals. The tolylene diisocyanate administration group (tdn) included 2 out of 3 individuals, and the 2,4-di-tert-butylphenol administration group (24b) included 3 out of 3 individuals. The cluster “E” includes one hydrazine monohydrate group (hmh) in three individuals and three 3-methoxy-3-methyl-1-butanol groups (mmb) in three individuals. It was. The cluster of “F” contained 1 of 3 individuals in the 2-isopropoxyethanol administration group (2ip) and 2 out of 3 individuals in the 4-ethylmorpholine administration group (4em). The cluster of “G” contained 1 of 3 individuals in the 2-isopropoxyethanol administration group (2ip) and 2 out of 3 individuals in the 2-butanone oxime administration group (2bo). The “H” cluster contained other samples including the control group. Moreover, all the chemical substances contained in the clusters “A” to “G” were known to have hepatotoxicity. This result means that these chemical substances can be distinguished from the control group by the expression variation pattern of the gene having the base sequence shown in SEQ ID NOs: 1 to 59, and the chemical substances can be classified.

  Next, the rat t-test was performed for the logarithmic transformation relative expression ratio of each gene by comparing the liver of a rat administered with repeated administration of 3,4-xylidine for 28 days with the liver of a control group administered with the solvent. The P value was calculated, and a gene group having an absolute value of the difference in the average value of the expression level between them of 1.0 or more and a P value of less than 0.001 was extracted. Gene group having a base sequence). Hierarchical cluster analysis was performed based on the expression variation pattern of the gene having the nucleotide sequence shown in SEQ ID NOs: 1-19. As a result, chemical substances could be roughly classified into 6 clusters. FIG. 3 shows the result of cluster analysis. The abbreviations in the figure are the same as those in FIG.

  In the cluster “A”, the tetrabromoethane administration group (tbe) was 3 out of 3 individuals, the o-dichlorobenzene administration group (dcb) was 3 out of 3 individuals, and the 3-cyanopyridine administration group (3cp) was 3 Three of the individuals, the 1,4-dibromobenzene-administered group (14d) was included in three of the three individuals, and the 3,4-xylidine-administered group (34x) was included in three of the three individuals. The “B” cluster contained 3 out of 3 individuals in the diheptyl phthalate administration group (dhp). In the cluster of “C”, N-methylaniline administration group (nma) is 3 out of 3 individuals, 3,5-xylidine administration group (35x) is 3 out of 3 individuals, 2-butanone oxime administration group (2bo) Were 3 out of 3 individuals, the 2-isopropoxyethanol administration group (2ip) was 1 in 3 individuals, and the 2,3-dimethylaniline administration group (23d) was included in 3 out of 3 individuals. The cluster of “D” included 1 out of 3 individuals in the 2-isopropoxyethanol administration group (2ip) and 2 out of 3 individuals in the hydrazine monohydrate administration group (hmh). In the cluster “E”, the sulfolane administration group (suf) is 3 out of 3 individuals, the p-cumylphenol administration group (pcp) is 3 out of 3 individuals, and the pseudocumene administration group (tmb) is 3 out of 3 individuals. The methacrylamide-administered group (mca) is one of three individuals, the p-ethylphenol-administered group (pep) is two of three individuals, the 1-bromo-3-chloropropane-administered group (bcp) is three of three individuals, The 1,3-dibromopropane administration group (13d) included 3 out of 3 individuals. The “F” cluster contained other samples including the control group. Moreover, the chemical substances contained in the clusters “A” to “G” were known to have hepatotoxicity except for one sample. This result means that these chemical substances can be distinguished from the control group and can be classified by the expression variation pattern of the gene having the nucleotide sequence shown in SEQ ID NOs: 1 to 19.

  Next, the rat t-test was performed for the logarithmic transformation relative expression ratio of each gene by comparing the liver of a rat administered with repeated administration of 3,4-xylidine for 28 days with the liver of a control group administered with the solvent. The P value was calculated, and a gene group having an absolute value of the difference in the average value of the expression levels of 1.0 or more and a P value of less than 0.0005 was extracted. Gene group having a base sequence). Hierarchical cluster analysis was performed based on the expression variation pattern of the gene having the base sequence shown in SEQ ID NOs: 1-13. As a result, chemical substances could be roughly classified into 6 clusters. FIG. 4 shows the results of cluster analysis. The abbreviations in the figure are the same as those in FIG.

  In the cluster “A”, the 3-cyanopyridine administration group (3cp) was 3 out of 3 individuals, the 1,4-dibromobenzene administration group (14d) was 3 out of 3 individuals, and the o-dichlorobenzene administration group (dcb ) Is 3 out of 3 individuals, 1-bromo-3-chloropropane administration group (bcp) is 2 out of 3 individuals, 1,3-dibromopropane administration group (13d) is 3 out of 3 individuals, tetrabromoethane administration group (Tbe) included 3 out of 3 individuals, and 3,4-xylidine administration group (34x) included 3 out of 3 individuals. The “B” cluster contained 3 out of 3 individuals in the diheptyl phthalate administration group (dhp). In the cluster of “C”, N-methylaniline administration group (nma) is 3 out of 3 individuals, 3,5-xylidine administration group (35x) is 3 out of 3 individuals, hydrazine monohydrate administration group (hmh) ) Included 2 out of 3 individuals, the 2-isopropoxyethanol administration group (2ip) included 3 out of 3 individuals, and the 2-butanone oxime administration group (2bo) included 3 out of 3 individuals. The cluster of “D” contained 3 out of 3 individuals in the 2,3-dimethylaniline administration group (23d). In the cluster “E”, the p-cumylphenol administration group (pcp) is 3 out of 3 individuals, the sulfolane administration group (suf) is 3 out of 3 individuals, the 1-bromo-3-chloropropane administration group (bcp) 1 of the 3 individuals, 2 of the 3 groups administered with p-ethylphenol (pep), and 3 of the 3 groups treated with pseudocumene (tmb). The “F” cluster contained other samples including the control group. Moreover, all the chemical substances contained in the clusters “A” to “E” were known to have hepatotoxicity. This result means that these chemical substances can be distinguished from the control group by the expression variation pattern of the gene having the base sequence shown in SEQ ID NOs: 1 to 13, and the chemical substances can be classified.

  The toxicity determining gene set of the present invention may be able to assist in monitoring liver toxicity, their diagnosis and / or determining the effectiveness of various measures or drugs against them.

Claims (33)

A method for evaluating the toxicity of a test chemical substance by measuring a gene expression level after exposing the test chemical substance to a living body or cells for a predetermined period,
(A) Prepare a plurality of cell samples derived from experimental animals or livers, and use a part of the test sample as the test sample after leaving the test chemical substance exposed to the test chemical substance for a predetermined period. Using as a reference sample a liver or liver-derived cell sample after treatment or exposure to said chemical solvent;
(B) About the said test | inspection sample, the expression level of the gene with respect to the arbitrary 1 or more selection biological response gene group selected from the biological response gene group as a gene group which has a base sequence shown by sequence number 1-74 is shown. A first gene expression level measurement step to measure,
(C) a second gene expression level measurement step for measuring the expression level of the gene for the selected biological response gene group for the reference sample;
(D) The gene expression levels measured in the first gene expression level measurement step and the second gene expression level measurement step are compared for each corresponding gene, and the test chemistry is based on the difference in the expression level of the gene. Evaluating the toxicity of the substance;
A method for evaluating the toxicity of a chemical substance, comprising:   2. The chemical substance toxicity evaluation method according to claim 1, wherein the biological response gene group is a gene group having a base sequence represented by SEQ ID NOs: 1 to 59.   The method for evaluating toxicity of a chemical substance according to claim 1, wherein the biological response gene group is a gene group having a base sequence represented by SEQ ID NOs: 1 to 19.   The method for evaluating toxicity of a chemical substance according to claim 1, wherein the biological response gene group is a gene group having a base sequence represented by SEQ ID NOs: 1 to 13.   The expression level of the gene is measured using an expression level in a reporter gene including a sequence encoding a reporter protein linked to a promoter sequence in each biological response gene in the biological response gene group as an index, The toxicity evaluation method for a chemical substance according to any one of claims 1 to 4.   A nucleic acid construct containing a reporter gene used in the method according to claim 5, a vector containing the nucleic acid construct, or a transformed cell having these introduced into a host cell, which is linked to the promoter sequence of the biological response gene A nucleic acid construct comprising a sequence encoding a reporter protein, a vector containing the nucleic acid construct, or a transformed cell having these introduced into a host cell.   The transformed host cell according to claim 6, wherein the host cell is an animal cell, a stem cell, or an embryonic stem cell. A method for determining and predicting the toxicity of a test chemical substance by detecting the influence of the chemical substance on the living body at the gene expression level,
(A) administering (exposing) a predetermined amount of a chemical substance known to have hepatotoxicity to a biological or liver-derived cell sample for a predetermined period;
(B) administering (exposing) a predetermined amount of a chemical substance known not to have liver toxicity to a cell sample derived from a living body or a liver for a predetermined period;
(C) administering (exposing) a predetermined amount to a cell sample derived from a living body or a liver for a predetermined period using the chemical solvent as a control;
(D) An mRNA is isolated from the liver of the living body or a cell sample derived from the liver, and any one or more selected from the group of biological response genes as a group of genes having the base sequences of SEQ ID NOs: 1 to 74 A measurement step for measuring a gene expression level with respect to a biological response gene;
(E) collecting the gene expression level together with the corresponding chemical substance, exposure amount, and exposure period as gene expression data;
(F) exposing a test chemical substance to a living or liver-derived cell sample at an appropriate concentration for a certain period of time;
(G) isolating mRNA from the living body-derived liver or the liver-derived cell sample, and measuring a gene expression level for the biological response gene selected in the step (D);
(H) collecting the gene expression level obtained in (G) as gene expression data together with the test chemical substance, the exposure amount and the exposure period;
(I) comparing the gene expression data collected in (H) with the corresponding gene expression data for verification collected in (E);
A method for evaluating the toxicity of a chemical substance, comprising:   9. The chemical substance toxicity evaluation method according to claim 8, wherein the biological response gene group is a gene group having a base sequence represented by SEQ ID NOs: 1 to 59.   The method for evaluating toxicity of a chemical substance according to claim 8, wherein the biological response gene group is a gene group having a base sequence represented by SEQ ID NOs: 1 to 19.   The method for evaluating toxicity of a chemical substance according to claim 8, wherein the biological response gene group is a gene group having a base sequence represented by SEQ ID NOs: 1 to 13.   The comparison of the gene expression data is a difference in gene expression level between a test chemical substance exposure group and a chemical substance exposure group that is known not to have hepatotoxicity. The chemical substance toxicity evaluation method according to any one of the above.   The comparison of the gene expression data is a cluster analysis using as an index the expression profile of the biological response gene group in a chemical substance exposure group and a chemical substance exposure group that is known not to have liver toxicity. The toxicity evaluation method for a chemical substance according to any one of claims 8 to 11.   Comparison of the gene expression data is characterized by using a correlation coefficient of an expression profile of the biological response gene group in a chemical substance exposure group and a chemical substance exposure group known to have no hepatotoxicity as an index. The toxicity evaluation method for a chemical substance according to any one of claims 8 to 11.   Chemicals known to have liver toxicity include 2-butanone oxime (CAS registry number 96-29-7), 3-cyanopyridine (CAS registry number 100-54-9), sulfolane (CAS registry number 126-33). -0), 2-isopropoxyethanol (CAS registration number 109-59-1), hydrazine monohydrate (CAS registration number 7803-57-8), 4-ethylmorpholine (CAS registration number 100-74-3) , 3-methoxy-3-methyl-1-butanol (CAS registration number 55539-66-3), o-dichlorobenzene (CAS registration number 95-50-1), 3,4-xylidine (CAS registration number 95-64) -7), N-methylaniline (CAS registration number 100-61-8), tolylene diisocyanate (CAS registration number 26471-62-5), 2- (dibutylamino) ethanol (CAS registration number 102-81-8) ), P-cumylphenol (CAS registration number 599-64-4), m-cresol (CAS registration number 108-39-4), 2,3-dimethylaniline (CAS registration number 87-59-2), N , N'-Dicyclohexylcarbodi Mido (CAS registration number 538-75-0), diheptyl phthalate (CAS registration number 3648-21-3), tetrabromoethane (CAS registration number 79-27-6), p-ethylphenol (CAS registration number 123- 07-9), 2,4-di-tert-butylphenol (CAS registration number 96-76-4), 3,5-xylidine (CAS registration number 108-69-0), 1,3-dibromopropane (CAS registration) 109-64-8), 1-bromo-3-chloropropane (CAS registration number 109-70-6), pseudocumene (CAS registration number 95-63-6), 1,4-dibromobenzene (CAS registration number 106- The method for evaluating toxicity of a chemical substance according to any one of claims 8 to 11, wherein the chemical substance is any one or more chemical substances selected from 37-6).   Chemicals known not to have liver toxicity include 2- (2-aminoethylamino) ethanol (CAS registration number 111-41-1), tetrahydrofurfuryl alcohol (CAS registration number 97-99-4), Methacrylamide (CAS registration number 79-39-0), ethyl trimethylammonium methacrylate (CAS registration number 5039-78-1), benzyltrimethylammonium chloride (CAS registration number 56-93-9), m-nitrobenzenesulfonic acid Sodium (CAS registration number 127-68-4), sodium 1-naphthylamine-4-sulfonate tetrahydrate (CAS registration number 130-13-2), dibutyl adipate (CAS registration number 105-99-7), N, N-dimethylbenzylamine (CAS registration number 103-83-3), n-hexadecane (CAS registration number 544-76-3), dicyclohexylamine (CAS registration number 101-83-7), and 2-amino- Select from 5-methylbenzenesulfonic acid (CAS registration number 88-44-8) The toxicity evaluation method for a chemical substance according to any one of claims 8 to 11, wherein the chemical substance toxicity evaluation method is any one or more chemical substances.   The gene expression level is measured by one of RT-PCR, Real Time PCR, iAFLP (Introduced Amplified Fragment Length Polymorphism), LAMP (Loop-Mediated Isothermal Amplification), nCounter Analysis system, and hybridization. The method for evaluating toxicity of a chemical substance according to any one of claims 1 to 16, wherein the method is used.   The chemical substance toxicity evaluation method according to claim 17, wherein the hybridization method is a microarray method or a blot method.   19. The chemical substance toxicity evaluation method according to claim 18, wherein the probe used in the microarray method or blotting method is a nucleotide or a protein.   20. The chemical substance toxicity evaluation method according to claim 19, wherein the nucleotide is mRNA, cDNA, or synthetic oligonucleotide.   21. The chemical substance toxicity evaluation method according to claim 19 or 20, wherein the nucleotide is a labeled nucleotide.   The measurement of the gene expression level is based on measurement of the presence or amount of a nucleic acid corresponding to the biological response gene or a protein encoded by the biological response gene. The toxicity evaluation method of the chemical substance as described in any one of these.   23. The chemical substance toxicity evaluation method according to claim 22, wherein the protein is measured by an immunological method.   24. The chemical substance according to claim 23, wherein the immunological method is based on a method of detecting an immunological complex of a specific antibody against a protein encoded by the biological response gene or a fragment thereof and a target protein. Toxicity evaluation method.   25. The chemical substance toxicity evaluation method according to claim 24, wherein the specific antibody is selected from a monoclonal antibody, a polyclonal antibody, a chimeric antibody, and an antibody fragment.   A chemical substance toxicity determination kit comprising a probe used in the method according to any one of claims 1 to 25, wherein the probe specifically hybridizes to the biological response gene or a transcription product thereof. A chemical toxicity evaluation kit comprising a molecule having a sequence of   27. The chemical substance toxicity evaluation kit according to claim 26, wherein the probe is a nucleotide or a protein.   28. The chemical toxicity evaluation kit according to claim 27, wherein the nucleotide is mRNA, cDNA, or a synthetic oligonucleotide.   29. The chemical substance toxicity evaluation kit according to claim 28, wherein the nucleotide hybridizes with a sense strand or an antisense strand of the biological response gene and has 10 to 100 bases.   30. The chemical substance toxicity evaluation kit according to claim 28 or 29, wherein the nucleotide is a labeled nucleotide.   31. The chemical substance toxicity evaluation kit according to claim 30, wherein the probe is a protein that is an antibody and / or an aptamer.   32. The chemistry according to claim 26, wherein the probe includes a DNA microarray, a DNA chip, a protein chip, or an antibody chip in which any one or more are immobilized on a solid support. Substance toxicity evaluation kit.   The chemical substance toxicity evaluation kit according to claim 32, wherein the solid support is glass, silicon, plastic, or a biological membrane.