JP2012196161A - Method of detecting biological effect of chemical substance using gene set - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of quickly and precisely detecting biological effects of a chemical substance using a gene set without using hematological or pathological techniques.SOLUTION: A method of determining and predicting the toxicity of a chemical substance includes: (A) providing a material to be tested to a plurality of experimental animals or liver cell samples only for a predetermined period of time to obtain testing samples, while using the rest as reference samples; (B) measuring the level of expression of one or more selective bioresponsive gene cluster selected from a group of bioresponsive genes having specific base sequences for both samples; and (C) evaluating hepatotoxicity that the chemical substance to be tested has based on differences in the level of gene expression between both samples.

Description

  The present invention relates to a method for detecting, diagnosing, predicting and / or treating an influence of a chemical substance on a living body, biotoxicity, 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 repeated dose toxicity tests mainly consist of hematological and histopathological examinations, and their biological information is limited. In addition, the evaluation by histopathological examination is easily influenced by the subjectivity of the person who made the decision, so that different expressions can be used for evaluating the toxicity between different chemical substances even though the same disease state is seen. There were few objective indicators.

  In addition, there are few examples of conducting gene expression fluctuation analysis on a large scale for exposure to multiple chemicals with hepatotoxicity, and identifying genes that change expression in vivo due to exposure to those chemical substances. The hepatotoxicity of chemical substances could not be evaluated.

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

  The present inventor has 9 genes whose expression was statistically significantly changed in the liver of the rat after repeated administration of tolylene diisocyanate (CAS registration number 26471-62-5) to the rat for 28 days. As a result, the present invention has been completed.

That is, the present invention provides the following.
1. A method for determining or predicting toxicity of a test chemical substance by measuring a gene expression level after the test chemical substance is exposed to a living body or a cell for a predetermined period,
(A) preparing a plurality of cell samples derived from an experimental animal or liver, giving a test sample with exposure to the test chemical substance for a part of the cell sample, and using the rest as a reference sample;
(B) About the said test sample, the expression level of the gene with respect to the at least 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 1-9 is measured. A first gene expression level measuring step to:
(B) 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;
(C) 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 levels of the genes. Assessing the hepatotoxicity of the substance;
A method for determining and predicting the toxicity of chemical substances characterized by comprising
2. 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, 2. The method for determining and predicting toxicity of a chemical substance according to 1 above.
3. A nucleic acid construct containing a reporter gene used in the method according to 2 above, a vector containing the nucleic acid construct, or a transformed cell having these introduced into a host cell,
A nucleic acid construct comprising a sequence encoding a reporter protein linked to a promoter sequence of the biological response gene, a vector containing the same, or a transformed cell in which these are introduced into a host cell.
4). 4. The transformed cell according to 3 above, wherein the host cell is an animal cell, a stem cell, or an embryonic stem cell.
5. The method further comprises: exposing the reference sample to a plurality of chemical substances including known chemical substances known to have hepatotoxicity or a solvent in which the chemical substances are dissolved for the predetermined period of time. Method for determining and predicting toxicity of chemical substances according to 1.
6). The known chemical substances are 2-butanone oxime (CAS registration number 96-29-7), 3-cyanopyridine (CAS registration number 100-54-9), sulfolane (CAS registration 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 56539-66-3), 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-cumyl Phenol (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'-dicyclohexylcarbodiimide (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 number 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 106-37-6) 6. The method for determining and predicting toxicity of a chemical substance according to the above 5, wherein the chemical substance is at least one chemical substance.
7). 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. 6. The method for determining and predicting toxicity of a chemical substance according to 1 or 5, wherein a method is used.
8). 8. The chemical substance toxicity discrimination / prediction method according to 7 above, wherein the hybridization method is a microarray method or a blot method.
9. 9. The chemical substance toxicity discrimination / prediction method according to 8 above, wherein the probe used in the microarray method or blotting method is a nucleotide or a protein.
10. 10. The toxicity determination / prediction method for a chemical substance according to 9, wherein the nucleotide is mRNA, cDNA, or synthetic oligonucleotide.
11. 11. The method for discriminating and predicting toxicity of a chemical substance according to the above 9 or 10, wherein the nucleotide is a labeled nucleotide.
12 The measurement of the gene expression level is based on the 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. Toxicity discrimination / prediction method for chemical substances listed.
13. 13. The chemical substance toxicity determination / prediction method according to the above item 12, wherein the protein is measured by an immunological method.
14 The toxicity of the chemical substance according to the above 13, 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. Discrimination / prediction method.
15. 15. The chemical substance toxicity discrimination / prediction method according to the above 14, wherein the specific antibody is selected from a monoclonal antibody, a polyclonal antibody, a chimeric antibody, and an antibody fragment.
16. A method of creating a gene expression database as reference data used to discriminate 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) 2-butanone oxime (CAS registration number 96-29-7), 3-cyanopyridine (CAS registration number 100-54-9), 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), sulfolane (CAS registration 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), ethyl trimethylammonium methacrylate (CAS registration) No. 5039-78-1), benzyltrimethylammonium chloride (CAS registration number 56-93-9), sodium m-nitrobenzenesulfonate (CAS registration number 127-68-4), sodium 1-naphthylamine-4-sulfonate Hydrate (CAS registration number 130-13-2), 3-methoxy-3-methyl-1-butanol (CAS registration) No. 56539-66-3), 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'-dicyclohexylcarbodiimide (CAS registration number 538-75-0) ), Diheptyl phthalate (CAS registration number 3648-21-3), tetrabromoethane (CAS registration number 79-27-6), dibutyl adipate (CAS registration number 105-99-7), 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), N, N-dimethyl Benzylamine (CAS registration number 103-83-3), 1 , 3-dibromopropane (CAS registration number 109-64-8), n-hexadecane (CAS registration number 544-76-3), 1-bromo-3-chloropropane (CAS registration number 109-70-6), pseudocumene ( CAS registry number 95-63-6), dicyclohexylamine (CAS registry number 101-83-7), 1,4-dibromobenzene (CAS registry number 106-37-6), and 2-amino-5-methylbenzenesulfone Administering (exposing) a predetermined amount of each of the acids (CAS Registry No. 88-44-8) to a living or liver-derived cell sample for a predetermined period;
(B) At least one or more selected from a gene group (biological response gene group) having a base sequence of SEQ ID NOs: 1 to 9 by isolating mRNA from the liver tissue of the living body or the cell sample derived from the liver A measurement step for measuring a gene expression level with respect to a biological response gene of
(C) database the gene expression level as a gene expression data together with the corresponding chemical substance, exposure amount and exposure period;
A gene expression database creation method for verification, comprising:
17. The measurement step uses 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 method for preparing a gene expression database for verification as described in 16 above.
18. 18. The method for creating a reference gene expression database according to 17, wherein the hybridization method is a microarray method or a blot method.
19. 19. The method for creating a reference gene expression database 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 creating a reference gene expression database as described in 19 above, wherein the nucleotide is mRNA, cDNA, or synthetic oligonucleotide.
21. 21. The method for creating a reference gene expression database as described in 19 or 20 above, wherein the nucleotide is a labeled nucleotide.
22. 17. The verification gene expression according to 16 above, wherein the measurement step is present by measuring a nucleic acid corresponding to the biological response gene or a protein encoded by the biological response gene. How to create a database.
23. 23. The method for creating a reference gene expression database according to the above 22, wherein the measurement is performed by an immunological method.
24. 24. The verification gene according to 23, wherein the immunological method is 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. How to create an expression database.
25. 25. The method for creating a gene expression database for verification according to 24, wherein the specific antibody is selected from a monoclonal antibody, a polyclonal antibody, a chimeric antibody, and an antibody fragment.
26. 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) 2-butanone oxime (CAS registration number 96-29-7), 3-cyanopyridine (CAS registration number 100-54-9), 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), sulfolane (CAS registration 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), ethyl trimethylammonium methacrylate (CAS registration) No. 5039-78-1), benzyltrimethylammonium chloride (CAS registration number 56-93-9), sodium m-nitrobenzenesulfonate (CAS registration number 127-68-4), sodium 1-naphthylamine-4-sulfonate Hydrate (CAS registration number 130-13-2), 3-methoxy-3-methyl-1-butanol (CAS registration) No. 56539-66-3), 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'-dicyclohexylcarbodiimide (CAS registration number 538-75-0) ), Diheptyl phthalate (CAS registration number 3648-21-3), tetrabromoethane (CAS registration number 79-27-6), dibutyl adipate (CAS registration number 105-99-7), 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), N, N-dimethyl Benzylamine (CAS registration number 103-83-3), 1 , 3-dibromopropane (CAS registration number 109-64-8), n-hexadecane (CAS registration number 544-76-3), 1-bromo-3-chloropropane (CAS registration number 109-70-6), pseudocumene ( CAS registry number 95-63-6), dicyclohexylamine (CAS registry number 101-83-7), 1,4-dibromobenzene (CAS registry number 106-37-6), and 2-amino-5-methylbenzenesulfone Administering (exposing) a predetermined amount of each of the acids (CAS Registry No. 88-44-8) to a living or liver-derived cell sample for a predetermined period;
(B) At least one or more selected from a gene group (biological response gene group) having a base sequence of SEQ ID NOs: 1 to 9 by isolating mRNA from the liver tissue of the living body or the cell sample derived from the liver A measurement step for measuring a gene expression level with respect to a biological response gene of
(C) collecting the gene expression level together with the corresponding chemical substance, exposure amount, and exposure period as gene expression data;
(D) exposing a test chemical substance to a living or liver-derived cell sample at an appropriate concentration for a certain period of time;
(E) isolating mRNA from the living body-derived liver tissue or the liver-derived cell sample, and measuring a gene expression level for the biological response gene selected in the step (B);
(F) collecting the gene expression level obtained in (E) as gene expression data together with the test chemical substance, the exposure amount and the exposure period;
(G) comparing the gene expression data collected in (F) with the corresponding gene expression data for matching collected in (C);
A method for determining and predicting the toxicity of chemical substances characterized by comprising
27. The measurement step uses one of RT-PCR, Real Time PCR, iAFLP (Introduced Amplified Fragment Length Polymorphism), LAMP (Loop-Mediated Isothermal Amplification), nCounter Analysis system, and hybridization. 27. A method for determining and predicting toxicity of a chemical substance according to the above item 26.
28. 28. The method for discriminating and predicting toxicity of a chemical substance according to 27, wherein the hybridization method is a microarray method or a blot method.
29. 29. The chemical substance toxicity discrimination / prediction method according to 28, wherein a probe used in the microarray method or blotting method is a nucleotide or a protein.
30. 30. The method for discriminating and predicting toxicity of a chemical substance according to the above 29, wherein the nucleotide is mRNA, cDNA, or synthetic oligonucleotide.
31. 31. The chemical substance toxicity discrimination / prediction method according to the item 29 or 30, wherein the nucleotide is a labeled nucleotide.
32. The determination of toxicity of a chemical substance according to 26, wherein the measuring step 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.・ Prediction method.
33. 33. The chemical substance toxicity determination / prediction method according to the item 32, wherein the measurement is performed by an immunological method.
34. 34. The chemical substance according to 33, wherein the immunological method is 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. Toxicity discrimination / prediction method.
35. 35. The method for determining and predicting toxicity of a chemical substance according to the above 34, wherein the specific antibody is selected from a monoclonal antibody, a polyclonal antibody, a chimeric antibody, and an antibody fragment.
36. 36. A chemical toxicity determination kit comprising a probe used in the method according to any one of 1 to 35 except 3 and 4, wherein the probe is the biological response gene or a transcription product thereof A chemical toxicity determination kit comprising a molecule having a sequence that specifically hybridizes to a chemical substance.
37. 36. The chemical substance toxicity determination kit according to 36, wherein the probe is a nucleotide or a protein.
38. 38. The chemical substance toxicity determination kit according to 37, wherein the nucleotide is mRNA, cDNA, or a synthetic oligonucleotide.
39. 40. The chemical substance toxicity determination kit according to 38, wherein the nucleotide hybridizes with a sense strand or an antisense strand of the biological response gene and has 10 to 100 bases.
40. 40. The chemical substance toxicity determination kit according to 38 or 39, wherein the nucleotide is a labeled nucleotide.
41. 37. The chemical substance toxicity determination kit according to 36, wherein the probe is a protein that is an antibody and / or an aptamer.
42. 44. The chemical substance according to any one of 36 to 41, wherein the probe includes a DNA microarray, a DNA chip, a protein chip, or an antibody chip in which at least one is fixed to a solid support. Toxicity discrimination kit.
43. 43. The chemical substance toxicity determination kit according to 42, wherein the solid support is glass, silicon, plastic, or a biological membrane.

  According to the present invention, it is possible to easily determine or predict whether or not a chemical substance is toxic to a living body by comparing gene expression patterns in the liver after the chemical substance is administered to the living body.

Hierarchical cluster analysis was performed based on the expression variation pattern of the gene having the base sequence shown in SEQ ID NO: 1-9. In the figure, “C1” is “water for injection used in the first experiment”, “2bo” is “2-butanone oxime”, “3cp” is “3-cyanopyridine”, “2ae” is “ 2- (2-aminoethylamino) ethanol ”,“ thf ”is“ tetrahydrofurfuryl alcohol ”,“ C2 ”is“ water for injection used in the second experiment ”,“ mca ”is“ methacrylamide ” `` Suf '' is `` sulfolane '', `` 2ip '' is `` 2-isopropoxyethanol '', `` hmh '' is `` hydrazine monohydrate '', `` 4em '' is `` 4-ethylmorpholine '', “C3” is “water for injection used in the third experiment”, “mta” is “ethyltrimethylammonium methacrylate”, “bac” is “benzyltrimethylammonium chloride”, “mns” is “m” -Sodium nitrobenzenesulfonate "," nat "is" 1-naphthylamine- “Sodium 4-sulfonate tetrahydrate”, “mmb” for “3-methoxy-3-methyl-1-butanol”, “C4” for “water for injection used in the fourth experiment”, “ `` dcb '' is `` o-dichlorobenzene '', `` 34x '' is `` 3,4-xylidine '', `` nma '' is `` N-methylaniline '', `` tdn '' is `` tolylene diisocyanate '', `` 2de "" (2- (dibutylamino) ethanol) "," C5 "means" olive oil used in the 5th experiment "," pcp "means" p-cumylphenol "," mcs "means" m- Cresol, 23d for 2,3-dimethylaniline, dhc for N, N'-dicyclohexylcarbodiimide, dhp for diheptyl phthalate, and C6 for No. “Olive oil” used in the 6th experiment, “tbe” for “tetrabromoethane”, “dba” for “dibutyl adipate”, “pep” for “P-E” “Lephenol”, “C7” “Olive oil used in the 7th experiment”, “24b” “2,4-di-tert-butylphenol”, “35x” “3,5-xylidine” `` Nda '' is `` N, N-dimethylbenzylamine '', `` 13d '' is `` 1,3-dibromopropane '', `` nhd '' is `` n-hexadecane '', `` C8 '' is `` sesame oil '' , `` Bcp '' is `` 1-bromo-3-chloropropane '', `` tmb '' is `` busoid cumene '', `` dha '' is `` dicyclohexylamine '', `` 14d '' is `` 1,4-dibromobenzene '', `` “ams” represents “2-amino-5-methylbenzenesulfonic acid”. In addition, the numbers attached to the abbreviations of the chemical substances represent the individual rats. The black and white bars indicate the presence or absence of liver toxicity. Black represents a chemical substance having liver toxicity, and white represents a chemical substance having no liver toxicity. Further, black circles and white circles represent samples to be compared when specifying genes having the nucleotide sequences shown in SEQ ID NOs: 1 to 9, black circles represent chemical substance administration samples, and white circles represent control samples. Yes. “A” to “E” represent clusters.

  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 be tested chemical substances (test groups) or their solvents (control groups). 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 9 specify a 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 an RNA molecule or the like.

  “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 cell” refers to an undifferentiated cell that has both the ability to self-replicate and the ability to produce a differentiated cell. Embryonic stem cell (ES cell), tissue stem cell, induced pluripotent hepatocyte (iPS) Examples include, but are not limited to, induced pluripotent stem cells).

  “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.

  “Hepatotoxicity” means acute or chronic liver failure or dysfunction that can be induced by a process of many diseases or disorders.

  “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.

  The “sample” preferably includes liver biopsy material and any body 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 at least one nucleotide or cDNA 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 tolylene diisocyanate (CAS registration number 26471-62-5) and male Sprague Dawley rat (6 weeks old) (Charles Japan). -This is a group of genes whose expression level has changed significantly in the liver by repeated administration to 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.

  37 kinds of chemical substances 2-butanone oxime, 3-butane oxime registered in the existing chemical substance toxicity database (http://dra4.nihs.go.jp/mhlw_data/jsp/SearchPage.jsp) of National Institute of Health Sciences Cyanopyridine, 2- (2-aminoethylamino) ethanol, tetrahydrofurfuryl alcohol, methacrylamide, sulfolane, 2-isopropoxyethanol, hydrazine monohydrate, 4-ethylmorpholine, ethyltrimethylammonium methacrylate chloride, benzyl chloride Trimethylammonium, sodium m-nitrobenzenesulfonate, sodium 1-naphthylamine-4-sulfonate, 3-methoxy-3-methyl-1-butanol, o-dichlorobenzene, 3,4-xylidine, N-methyl Aniline, 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-methylbenzene Sulfonic 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.

  As an experiment schedule, the repeated administration test of 37 kinds of chemical substances was conducted in 8 times (see Table 1). In the table, "1st" to "8th" represent experimental times. For example, in the first experiment, 2-butanone oxime, 3-cyanopyridine, 2- (2-aminoethylamino) It shows that the experiment was conducted at the same time using ethanol, tetrahydrofurfuryl alcohol, and water for injection as a solvent thereof as a control group. Moreover, the 1st time-the 8th time have shown that the time of experiment differs, respectively. Furthermore, in the column of “abbreviation” in the table, the abbreviation of the chemical substance used in the application documents in the present invention is described, and in the “CAS registration number” column, the CAS of each chemical substance is described. The registration number is described.

  For the chemical administration solution, 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 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 , 300 mg / kg / day for tolylene diisocyanate, 250 mg / kg / day for 2- (dibutylamino) ethanol, 700 mg / kg / day for p-cumylphenol, 1,000 mg / kg / day for m-cresol, 2 , 3-dimethylaniline 200 mg / kg / day, N, N'-dicyclohexylcarbodiimide 300 mg / kg / day, diheptyl phthalate 1,000 mg / kg / day, tetrabromoethane 200 mg / kg / day, dibutyl adipate 1,000 mg / kg / day, p-ethylphenol 1,000 mg / kg / day, 2,4-di-tert-butylphenol 300 mg / kg / day, 3,5-xylidine 200 mg / kg / day, 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 Is 1,000 mg / kg / day, dicyclohexylamine is 70 mg / kg / day, 1,4-dibromobenzene is 300 mg / kg / day, and 2-amino-5-methylbenzenesulfonic acid is 1,000 mg / kg / day. Calculate the dosing solution volume from the measured weight of the rat as the subject of administration, 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 tissue 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 the synthetic DNA for microarray. The production method and conditions of the microarray are not limited. For example, the production method described in (Schena, M. et. Al., Science, 270, 467-470. (1995)) can be used.

  A rat gene fragment library (manufactured by Microdiagnostic) was printed on a slide glass (manufactured by Matsunami Glass Industrial Co., Ltd., HA-coated slide glass) using a micro-volume dispensing device (manufactured by Microdiagnostic) to produce 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.

<Statistical processing>
In order to select a gene useful for discriminating the effects of chemical substances on the living body, liver and water for injection were administered to rats that were repeatedly administered tolylene diisocyanate (CAS registration number 26471-62-5) for 28 days. Compared with the liver of a control group rat, Student's t-test was performed for the logarithmic transformation relative expression ratio of each gene to calculate the P value. Nine genes, which are gene groups in which the difference in the average expression level between the chemical substance administration group and the control group was more than doubled and the P value was less than 0.01, were identified. Next, the 9 genes were extracted from the gene expression profiles obtained from the livers of rats administered with the remaining 36 kinds of chemical substances and their respective controls (Tables 2 to 16). In the table, the “SEQ ID NO” column indicates the SEQ ID NO of the identified gene. In addition, the abbreviations in the table use those described in Table 1, and the numbers accompanying the abbreviations represent the individual.

<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 NO: 1-9. 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, five large clusters (A to E) were formed. Cluster A includes 1,4-dibromobenzene (represented by “14d”), 1,3-dibromopropane (represented by “13d”), 3-cyanopyridine (represented by “3cp”), 3, 4-Xylidine (represented by “34x”), tetrabromoethane (represented by “tbe”), o-dichlorobenzene (represented by “dcb”) and 1-bromo-3-chloropropane (represented by “bcp”) Cluster B contains diheptyl phthalate (represented by “dhp”), and cluster C comprises 2 of 3 individuals of buoyscumene (represented by “tmb”). , 3,5-xylidine (represented by “35x”) and 2,3-dimethylaniline (represented by “23d”), and cluster D contains p-cumylphenol (represented by “pcp”). 2) out of 3 individuals of 2,4-di-tert-butylphenol (represented by “24b”), sulfolane (represented by “suf”) 2 of the individuals, 1 of 3 individuals of pseudocumene (represented by “tmb”), N-methylaniline (represented by “nma”) and tolylene diisocyanate (represented by “tdn”) are included. Cluster E contained the remaining chemicals including the control group (FIG. 1). Clusters A to E contained 15 kinds of chemical substances known to have hepatotoxicity in addition to tolylene diisocyanate which is a chemical substance used for specifying the gene (9 genes). .

  These results show that by comparing the gene expression variation pattern of the gene set having the base sequences shown in SEQ ID NOs: 1 to 9, a chemical substance that has a high probability of having certain effects on the control group and certain liver toxicity It is suggested that it is possible to discriminate.

  The liver toxicity determination gene set of the present invention may be able to help determine liver damage, ie liver disease, injury or toxicity monitoring, their diagnosis and / or the effectiveness of various measures or drugs against them There is sex.

Claims (43)

A method for determining or predicting toxicity of a test chemical substance by measuring a gene expression level after the test chemical substance is exposed to a living body or a cell for a predetermined period,
(A) preparing a plurality of cell samples derived from an experimental animal or liver, giving a test sample with exposure to the test chemical substance for a part of the cell sample, and using the rest as a reference sample;
(B) About the said test sample, the expression level of the gene with respect to the at least 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 1-9 is measured. A first gene expression level measuring step to:
(B) 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;
(C) 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 levels of the genes. Assessing the hepatotoxicity of the substance;
A method for determining and predicting the toxicity of chemical substances characterized by comprising   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 determination / prediction method for a chemical substance according to claim 1. A nucleic acid construct containing a reporter gene used in the method according to claim 2, a vector containing the same, or a transformed cell having these introduced into a host cell,
A nucleic acid construct comprising a sequence encoding a reporter protein linked to a promoter sequence of the biological response gene, a vector containing the same, or a transformed cell in which these are introduced into a host cell.   4. The transformed cell according to claim 3, wherein the host cell is an animal cell, a stem cell, or an embryonic stem cell.   The method further comprises: exposing the reference sample to a plurality of chemical substances including known chemical substances known to have hepatotoxicity or a solvent in which the chemical substances are dissolved for the predetermined period of time. Item 1. A method for determining and predicting the toxicity of a chemical substance according to Item 1.   The known chemical substances are 2-butanone oxime (CAS registration number 96-29-7), 3-cyanopyridine (CAS registration number 100-54-9), sulfolane (CAS registration 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 56539-66-3), 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-cumyl Phenol (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'-dicyclohexylcarbodiimide (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 number 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 106-37-6) The chemical substance toxicity determination / prediction method according to claim 5, wherein the chemical substance toxicity determination / prediction method is at least one chemical substance.   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. 6. The chemical substance toxicity determination / prediction method according to claim 1 or 5, wherein the method is used.   8. The chemical substance toxicity determination / prediction method according to claim 7, wherein the hybridization method is a microarray method or a blot method.   9. The chemical substance toxicity discrimination / prediction method according to claim 8, wherein the probe used in the microarray method or blotting method is a nucleotide or a protein.   10. The chemical substance toxicity discrimination / prediction method according to claim 9, wherein the nucleotide is mRNA, cDNA, or synthetic oligonucleotide.   11. The chemical substance toxicity determination / prediction method according to claim 9, wherein the nucleotide is a labeled nucleotide.   6. 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. Toxicity discrimination / prediction method for chemical substances described in 1.   13. The chemical substance toxicity determination / prediction method according to claim 12, wherein the protein is measured by an immunological method.   14. The chemical substance according to claim 13, wherein the immunological method is 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. Toxicity discrimination / prediction method.   15. The chemical substance toxicity discrimination / prediction method according to claim 14, wherein the specific antibody is selected from a monoclonal antibody, a polyclonal antibody, a chimeric antibody, and an antibody fragment. A method of creating a gene expression database as reference data used to discriminate 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) 2-butanone oxime (CAS registration number 96-29-7), 3-cyanopyridine (CAS registration number 100-54-9), 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), sulfolane (CAS registration 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), ethyl trimethylammonium methacrylate (CAS registration) No. 5039-78-1), benzyltrimethylammonium chloride (CAS registration number 56-93-9), sodium m-nitrobenzenesulfonate (CAS registration number 127-68-4), sodium 1-naphthylamine-4-sulfonate Hydrate (CAS registration number 130-13-2), 3-methoxy-3-methyl-1-butanol (CAS registration) No. 56539-66-3), 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'-dicyclohexylcarbodiimide (CAS registration number 538-75-0) ), Diheptyl phthalate (CAS registration number 3648-21-3), tetrabromoethane (CAS registration number 79-27-6), dibutyl adipate (CAS registration number 105-99-7), 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), N, N-dimethyl Benzylamine (CAS registration number 103-83-3), 1 , 3-dibromopropane (CAS registration number 109-64-8), n-hexadecane (CAS registration number 544-76-3), 1-bromo-3-chloropropane (CAS registration number 109-70-6), pseudocumene ( CAS registry number 95-63-6), dicyclohexylamine (CAS registry number 101-83-7), 1,4-dibromobenzene (CAS registry number 106-37-6), and 2-amino-5-methylbenzenesulfone Administering (exposing) a predetermined amount of each of the acids (CAS Registry No. 88-44-8) to a living or liver-derived cell sample for a predetermined period;
(B) At least one or more selected from a gene group (biological response gene group) having a base sequence of SEQ ID NOs: 1 to 9 by isolating mRNA from the liver tissue of the living body or the cell sample derived from the liver A measurement step for measuring a gene expression level with respect to a biological response gene of
(C) database the gene expression level as a gene expression data together with the corresponding chemical substance, exposure amount and exposure period;
A gene expression database creation method for verification, comprising:   The measurement step uses 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 creating a reference gene expression database according to claim 16.   The method for creating a reference gene expression database according to claim 17, wherein the hybridization method is a microarray method or a blot method.   The method for creating a reference gene expression database according to claim 18, wherein the probe used in the microarray method or blotting method is a nucleotide or a protein.   The method for creating a reference gene expression database according to claim 19, wherein the nucleotide is mRNA, cDNA, or synthetic oligonucleotide.   21. The method for creating a reference gene expression database according to claim 19 or 20, wherein the nucleotide is a labeled nucleotide.   17. The verification gene according to claim 16, wherein the measuring step is present by measuring the nucleic acid corresponding to the biological response gene or the protein encoded by the biological response gene. How to create an expression database.   The method for creating a reference gene expression database according to claim 22, wherein the measurement is performed by an immunological method.   24. The collating method according to claim 23, wherein the immunological method is 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. How to create a gene expression database.   The method for creating a reference gene expression database 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 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) 2-butanone oxime (CAS registration number 96-29-7), 3-cyanopyridine (CAS registration number 100-54-9), 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), sulfolane (CAS registration 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), ethyl trimethylammonium methacrylate (CAS registration) No. 5039-78-1), benzyltrimethylammonium chloride (CAS registration number 56-93-9), sodium m-nitrobenzenesulfonate (CAS registration number 127-68-4), sodium 1-naphthylamine-4-sulfonate Hydrate (CAS registration number 130-13-2), 3-methoxy-3-methyl-1-butanol (CAS registration) No. 56539-66-3), 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'-dicyclohexylcarbodiimide (CAS registration number 538-75-0) ), Diheptyl phthalate (CAS registration number 3648-21-3), tetrabromoethane (CAS registration number 79-27-6), dibutyl adipate (CAS registration number 105-99-7), 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), N, N-dimethyl Benzylamine (CAS registration number 103-83-3), 1 , 3-dibromopropane (CAS registration number 109-64-8), n-hexadecane (CAS registration number 544-76-3), 1-bromo-3-chloropropane (CAS registration number 109-70-6), pseudocumene ( CAS registry number 95-63-6), dicyclohexylamine (CAS registry number 101-83-7), 1,4-dibromobenzene (CAS registry number 106-37-6), and 2-amino-5-methylbenzenesulfone Administering (exposing) a predetermined amount of each of the acids (CAS Registry No. 88-44-8) to a living or liver-derived cell sample for a predetermined period;
(B) At least one or more selected from a gene group (biological response gene group) having a base sequence of SEQ ID NOs: 1 to 9 by isolating mRNA from the liver tissue of the living body or the cell sample derived from the liver A measurement step for measuring a gene expression level with respect to a biological response gene of
(C) collecting the gene expression level together with the corresponding chemical substance, exposure amount, and exposure period as gene expression data;
(D) exposing a test chemical substance to a living or liver-derived cell sample at an appropriate concentration for a certain period of time;
(E) isolating mRNA from the living body-derived liver tissue or the liver-derived cell sample, and measuring a gene expression level for the biological response gene selected in the step (B);
(F) collecting the gene expression level obtained in (E) as gene expression data together with the test chemical substance, the exposure amount and the exposure period;
(G) comparing the gene expression data collected in (F) with the corresponding gene expression data for matching collected in (C);
A method for determining and predicting the toxicity of chemical substances characterized by comprising   The measurement step uses one of RT-PCR, Real Time PCR, iAFLP (Introduced Amplified Fragment Length Polymorphism), LAMP (Loop-Mediated Isothermal Amplification), nCounter Analysis system, and hybridization. 27. The method for discriminating and predicting toxicity of a chemical substance according to claim 26.   28. The chemical substance toxicity determination / prediction method according to claim 27, wherein the hybridization method is a microarray method or a blot method.   29. The chemical substance toxicity discrimination / prediction method according to claim 28, wherein the probe used in the microarray method or blotting method is a nucleotide or a protein.   30. The chemical substance toxicity discrimination / prediction method according to claim 29, wherein the nucleotide is mRNA, cDNA, or synthetic oligonucleotide.   31. The chemical substance toxicity determination / prediction method according to claim 29 or 30, wherein the nucleotide is a labeled nucleotide.   27. The toxicity of a chemical substance according to claim 26, wherein the measuring step is performed by measuring a nucleic acid corresponding to the biological response gene or a protein encoded by the biological response gene. Discrimination / prediction method.   33. The chemical substance toxicity determination / prediction method according to claim 32, wherein the measurement is performed by an immunological method.   The chemical substance according to claim 33, wherein the immunological method is 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 discrimination / prediction method.   35. The chemical substance toxicity discrimination / prediction method according to claim 34, wherein the specific antibody is selected from a monoclonal antibody, a polyclonal antibody, a chimeric antibody, and an antibody fragment. A chemical toxicity determination kit including a probe used in the method according to any one of claims 1 to 35, excluding claims 3 and 4,
A chemical substance toxicity determination kit, wherein the probe includes a molecule having a sequence that specifically hybridizes to the biological response gene or a transcription product thereof.   37. The chemical substance toxicity determination kit according to claim 36, wherein the probe is a nucleotide or a protein.   38. The chemical toxicity determination kit according to claim 37, wherein the nucleotide is mRNA, cDNA, or a synthetic oligonucleotide.   39. The chemical substance toxicity determination kit according to claim 38, wherein the nucleotide hybridizes with a sense strand or an antisense strand of the biological response gene and has 10 to 100 bases.   40. The chemical substance toxicity determination kit according to claim 38 or 39, wherein the nucleotide is a labeled nucleotide.   37. The chemical toxicity determination kit according to claim 36, wherein the probe is a protein that is an antibody and / or an aptamer.   The chemical substance according to any one of claims 36 to 41, wherein the probe includes a DNA microarray, a DNA chip, a protein chip, or an antibody chip in which at least one or more is fixed to a solid support. Toxicity discrimination kit.   The chemical substance toxicity determination kit according to claim 42, wherein the solid support is glass, silicon, plastic, or a biological membrane.