WO2005024020A1 - Method of screening carcinogenecity of chemical - Google Patents

Abstract

It is intended to provide a method of screening carcinogenicity of a chemical which can be conveniently and highly reproducibly carried out at a lower cost within a short period of time; and a microarray and a gene set appropriately usable in screening carcinogenicity of a chemical as described above. The expression amount of a liver cancer-associated gene, from among genes involved in mammalian genome, is detected. More specifically speaking, a chemical having known carcinogenicity and another chemical having unknown carcinogenicity are administered to mammals or their liver cells are treated by these chemicals. Then mRNA is extracted from vital liver cells of mammals of the chemical-treated group and mammals of the untreated group and the expression amount of the target gene is analyzed. Based on the results of the analysis, clustering analysis is further carried out to thereby screen the carcinogenicity of the chemical having unknown carcinogenicity as described above. A gene set comprising a probe hybridizable with the base sequence of mRNA transcribed from the target gene as described above or a sequence complementary to the mRNA as described above. A microarray having this probe loaded thereon.

Description

 TECHNICAL FIELD The present invention relates to a method for screening carcinogenicity of a chemical substance, and a microarray and a gene set used for a method of screening for carcinogenesis of the chemical substance.

'' Background technology

 To evaluate the safety of various newly synthesized chemicals, guidelines for various toxicity tests have been developed and implemented. Major examples of toxicity tests include mutagenicity tests and carcinogenicity tests.

 Experimentally predicting the carcinogenicity of a chemical in a toxicity test is an extremely important issue in considering the effect of the chemical on the human body. As a powerful test method for examining the carcinogenicity of a chemical substance, for example, there is a carcinogenicity test using experimental animals such as rats. This is because a chemical substance is administered to experimental animals such as rats for several months to several years, and carcinogenesis of the administered chemical substance is determined by detecting the occurrence of cancer in the experimental animal to which the chemical substance is administered. (For example, see the following document).

 Ito et al., “Toxicity Test Course 13 Carcinogenicity” Jinjinshokan, June 1991, p. 1-210.

 Disclosure of the invention

 However, in the conventional method of administering a chemical substance to an experimental animal to examine the carcinogenicity of the histological substance, a very long time of several months to several years, a large amount of experimental cost, Skilled scholars are required.

Therefore, an object of the present invention is to provide a method of screening for carcinogenicity of a chemical substance, which can be carried out easily and with good reproducibility in a short time at lower cost. It is another object of the present invention to provide a microarray and a gene set that can be suitably used when screening the carcinogenicity of a chemical substance. In order to solve a powerful problem, the present inventors conducted a study to test the carcinogenicity of a chemical substance by focusing on liver cancer that typically occurs first due to the ingested chemical substance. It is expected that several to several hundred genes whose expression levels fluctuate biologically significantly will be involved in the development of liver cancer.

 That is, the present invention provides a carcinogenic screening method for a chemical substance, which detects the expression level of a gene associated with menstrual cancer among the genes included in the mammalian genome.

 The present invention also provides a method for screening for carcinogenicity of a chemical substance, comprising: administering a chemical substance having a known carcinogenicity and a chemical substance having an unknown carcinogenicity to a mammal or causing it to act on a hepatocyte thereof; Screening for the carcinogenicity of the unknown chemical by detecting the expression level of genes related to liver cancer in hepatocytes of mammals in the untreated group and performing clustering analysis based on the detection results The present invention provides a method for screening for a carcinogenic substance.

 Further, the present invention provides a method for screening for a carcinogenicity of a chemical substance, wherein the mammal is any one of a human, a mouse, and a rat.

Furthermore, the present invention provides a method for screening for carcinogenicity of such a chemical substance, wherein the expression level of at least one gene is detected among a total of 240 genes shown in Tables 1, 2, and 3 below. The present invention provides a method for screening carcinogenic substances.

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80 £ contract OO dfAI W 0Z0 simple SOOZ OAV Furthermore, the present invention provides a method for screening for carcinogenicity of such a chemical substance, wherein the expression level of at least 20 of the total 240 genes shown in Tables 1, 2 and 3 is detected. The present invention provides a method for screening carcinogenic substances.

 Further, the present invention provides a method for screening for carcinogenicity of such a chemical substance, wherein the expression level of at least 50 kinds of genes out of a total of 240 kinds of genes shown in Tables 1, 2 and 3 above is determined. Provided is a carcinogenic screening method for a chemical substance to be detected.

Furthermore, the present invention provides a method of screening for carcinogenicity of such a chemical substance, wherein the expression level of at least one gene is detected among a total of 125 genes shown in Tables 4 and 5 below. A method for screening for a carcinogenicity of a chemical substance is provided.

Table 4

ACC # Gene No. ACCt Gene

NM— 012488 A2m 41 N_1 5878 Fapb5 Drawing 52 o; 2u-globulin 1 030832 Fabp7

NM 1 031760 Abcbl 1 X05834 FBN

NM— 012891 Acadvl N J12559 Fgg

NM_134432 Agt AF01 828 Figf

N .012898 Ahsg NM-012792 Fmol

NM— 031010 Alox12 NM— 024127 Gadd45a one 012738 Apoal NM— 017251 Gjb1

NM_0131 12 Apoa2 NM_019630 Gludins

N J12737 Apoa4 NM-012571 Go "

NM_012501. Apoc3 51 NM— 013177 6ot2

NM J 38828 Apoe NM_030826 Gpx1

NM-1 017134 Arg1 N _017013 Gsta2

M36708 Ass NM— 177426 Gstm2

NM—030850 Bhmt NM J 38974 Gstp2

NM.022399 Calr NM 053448 Hdac3

NM— 012727 Camk4 N _022179 Hk3

NM-1 012520 Cat M33648 H G-CoA

NM_053021 Clu N _013134 Hmgcr

NM-1 017202 Cox4a NM— 017080 Hsd1 1 b1

NM— 053586 Cox5b 61 NM_024392 Hsd17b4

NM_019360 Cox6c S45392 HSP90-beta

NM_017096 Crp NM 1 013060 Id2

U22893 Csda MM— 013144 Igfbpl

N _031315 Cte1 S58528 INTA

NM_013156 Ctsl NM— 031768 Itgae

NM— 012839 Cycs NM_019369 Itih4

Μ1Ϊ251 Cyp2b1 N _017138 Lamrl

NM—01 strokes Cyp2c1 1 N _022196 Lif

N _031839 Cyp2c23 N _013136 ak

K03501 Cyp2c9 71 N _031643 Map2kl image 161 Cyp3a1 NM J 33283 Map2k2

MM-1 012942 Cyp7a1 N _017246 ap2k5

NM-I 022513 0 / T-ST Tsuyoshi 053887 Map3k1

NM— 031853 Dbi NM— 053842 Mapkl

NM—138877 Dial NM_017347 Mapk3

NM— 053354 Dnmtl N _031622 apk6

NM-1 017245 Eef2 NM_021846 'cl1

NM— 053849 Erp70 NM-1 022673 Mecp2

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8UGQ # οον • ON 9U89 # D0V • O s 挲 ClO / t ^ OOZdf / X3 <I 0Z0 丽 OAV Furthermore, the present effort provides a method of screening for the carcinogenicity of such chemicals, which detects the expression level of at least 20 of the 125 total genes shown in Tables 4 and 5 above. A method of carcinogenic screening of a substance is provided. Further, the present invention provides a method for screening for the carcinogenicity of such a chemical substance, which comprises detecting the expression level of at least 50 kinds of genes out of a total of 125 kinds of genes shown in Tables 4 and 5 above. A method of carcinogenic screening of a substance is provided. Further, the present invention relates to a method for screening for the carcinogenicity of a chemical substance, wherein the carcinogenicity screening method for the chemical substance for detecting the expression levels of all 125 kinds of genes shown in Tables 4 and 5 above is used. Provide a method of

 Furthermore, the present invention provides a microarray equipped with a probe that hybridizes with a nucleotide sequence of mRNA transcribed from a gene associated with liver cancer among genes included in the genome of a mammalian animal, or a complementary sequence of the mRNA. I will provide a.

 Further, the present invention provides the microarray, wherein the mammal is any one of a human, a mouse, and a rat.

 Further, the present invention provides the microarray on which a plurality of types of probes are mounted, wherein each probe has at least one gene among a total of 240 types of genes shown in Tables 1, 2, and 3 above. The present invention provides a microarray that hybridizes with the nucleotide sequence of mRNA transcribed from or the complementary sequence of mRNA.

Further, the present invention relates to the microarray having at least 20 kinds of probes, wherein each probe has at least one of a total of 240 kinds of genes shown in Tables 1, 2 and 3 above. The present invention provides a microarray that hybridizes with a nucleotide sequence of mRNA transcribed from two genes or a complementary sequence of the mRNA. Furthermore, the present invention relates to the microarray having at least 50 kinds of probes, wherein each probe has at least one of a total of 240 kinds of genes shown in Tables 1, 2, and 3 above. The present invention provides a microarray that hybridizes with a nucleotide sequence of mRNA transcribed from two genes or a complementary sequence of the mRNA. Furthermore, the present invention relates to the microarray equipped with a plurality of types of probes, wherein each probe is transcribed from at least one gene out of a total of 125 types of genes shown in Tables 4 and 5 above. The nucleotide sequence of mRNA, or the complement of mRNA A microarray that hybridizes to the sequence is provided.

 Furthermore, the present invention provides the microarray, wherein at least 20 types of probes are mounted, wherein each probe is transcribed from at least one of a total of 125 types of genes shown in Tables 4 and 5 above. And a microarray that hybridizes with the nucleotide sequence of mRNA or the complementary sequence of the mRNA.

 Furthermore, the present invention provides the microarray, wherein at least 50 types of probes are mounted, wherein each probe is transcribed from at least one gene of a total of 125 types of genes shown in Tables 4 and 5 above. And a microarray that hybridizes with the nucleotide sequence of the mRNA or the complementary sequence of the mRNA.

 Further, the present invention provides the microphone-mouth array on which at least 125 types of probes are mounted, wherein each probe has an mRNA transcribed from at least one of a total of 125 types of genes shown in Tables 4 and 5. A microarray that hybridizes with a base sequence or a complementary sequence of the mRNA is provided.

 Further, the present effort provides a gene set consisting of probes mounted on such a microarray.

 Furthermore, the present invention provides a screening method for carcinogenicity of chemical substances, wherein the method for screening for carcinogenicity of chemical substances described above is detected using a method comprising the following steps:

 (1) a step of preparing a nucleic acid labeled as type II using mRNA extracted from hepatocytes;

 (2) contacting the labeled nucleic acid with the probe in a three-dimensional microarray having a probe that hybridizes with a nucleotide sequence of mRNA transcribed from a target gene or a complementary sequence of the mRNA;

 (3) a step of detecting a signal strength that changes based on the binding strength between the labeled nucleic acid and the probe.

Furthermore, the present invention provides a probe DNA set consisting of at least two of the DNAs of SEQ ID NOs: 1 to 126, or DNAs complementary to any of the above; l to 10 mer, the length of the DNA is 11 to 2 Omer, and the length of the DNA is 21 to 3 O mer, the length of the DNA is 31 to 4 O mer, the length of the DNA is 41 to 5 O mer, and Also provided are those with a DNA length of 51-6 O mer. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic configuration diagram showing a three-dimensional microarray system used for the method of screening for carcinogenicity of a chemical substance of the present invention.

 FIG. 2 is an enlarged schematic cross-sectional view of a part of the three-dimensional microarray system shown in FIG. '

 FIG. 3 is a schematic diagram showing the result of clustering analysis in the method of screening for carcinogenicity of a chemical substance of the present invention.

 FIG. 4 is a schematic diagram showing the results of clustering analysis in the method for screening carcinogenicity of a chemical substance of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

<Screening method for carcinogenicity of chemical substances>

 The method for screening carcinogenicity of a chemical substance of the present invention is characterized by detecting the expression level of a gene associated with liver cancer among the genes included in the genome of mammals. The liver is the first place where the effects of a chemical typically appear, from the digestive and absorption pathways of the intake of the chemical. That is, if the possibility of the occurrence of liver cancer can be predicted, it becomes an index of the effect of the chemical substance on the living body. In addition, by detecting the expression level of genes related to liver cancer, it is possible to predict the carcinogenicity of a chemical substance in a shorter period of time and at lower cost than in the conventional method of detecting cancer pathologically. be able to.

 It is preferable to carry out the specific method for carrying out the carcinogenic screening method of the chemical substance of the present invention, for example, as follows.

First, a chemical known to be carcinogenic is administered to a mammal or acts on its hepatocytes. In the following, administer the chemical substance to mammals or cause them to act on hepatocytes This is called processing. It is preferable to treat at least three animals per group with one chemical substance, and to use the average value in gene expression level analysis and clustering analysis described below. This is to reduce data variability due to individual differences between animals. In principle, one animal is treated with one chemical substance, but this is not the case when performing carcinogenicity screening with multiple chemical substances.

 It is also possible to investigate the carcinogenic dose relationship for a single chemical. In this case, a plurality of treatment groups for one chemical substance with different doses or effects are provided.

 By doing so, the method of screening for carcinogenicity of a chemical substance of the present invention can be applied to the purpose of detecting what dose and how much carcinogenicity a given chemical substance has.

 The mammal is preferably a human, a mouse, or a rat. For these animals, construction of a database on genetic information is relatively advanced among mammals.

 In addition, it is preferable that the treated animal be in a very early period after weaning, and in the case of a rat, for example, it is desirable that the animal be 6 weeks or older. If the age is more than 6 weeks, adverse effects such as the appearance of various effects other than the chemical substance to be treated may occur.

 As a method of administering a chemical substance to a mammal, there are, specifically, a method of ingesting the chemical substance in a feed and a method of directly administering the substance to the intraperitoneal cavity. When a chemical substance is mixed and administered to the feed, it is preferable to mix the chemical substance so that the concentration of the chemical substance is 5% or less in consideration of nutritional viewpoints. As a method of causing a chemical substance to act on moon cells of the mammal, for example, a liver tissue or the like may be isolated and cultured from a living body, and the chemical substance may directly act on the cultured cell tissue.

In conventional carcinogenicity tests, carcinogenicity could not be detected until the pathological manifestation of cancer became carcinogenic, which required several months to several years of experimental time. Since the amount of mRNA resulting from the expression of γ is examined, the administration period can be greatly reduced. When mixed with the above-mentioned feed and fed to the test animal, for example, it may be one to two weeks for rats, and when administered directly intraperitoneally, For example, for rats, 1-2 weeks may be sufficient. When a chemical substance is directly applied to cultured hepatocytes, the carcinogenicity can be screened in an extremely short time, such as 24 hours, because of the high sensitivity to the chemical substance.

The chemical substances whose carcinogenicity is known are classified into substances that induce carcinogenicity and substances that do not induce carcinogenicity. Examples of the former are diet hylnitros ami ne (DEN), 2-amino—3,8—d ime t hy li mi dazo [4, 5—f] quinoxaline (Me IQ) ^ henobarbita 1 (PB), clofibrate, 2—acetyl am inofluorene ^ acryl am ide, actin omy cin D, barbital, benzene, captan, cyclophosph am ide, dicofol, diet hy lstiibestrol, epichloro hy dri n¾ include throat force ^s, C as an example of the latter affeicacid, Ca techol, No bi 1 etin, G arcinol, Z er umb one, l - aceto xy c ii avico 1 acetate (a Ji a), acety丄salicyclicacid, aniline, anthracene, benz 0 in, busulfan, butylurethane, there capro l. actam, diazinon, d ime thoateethion ami de such as power ^s.

 Next, mRNA is extracted from hepatocytes, for example, by extracting an arbitrary part of the liver from a living body and extracting mRNA using a guanidine thiosinate method or the like. The expression level of the target gene is analyzed based on the extracted mRNA. Here, the target gene is a gene involved in liver cancer among the genes included in the mammalian genome. Will be described.

In the method of screening for carcinogenicity of a chemical substance of the present invention, it is preferable to detect the expression level of at least one gene among a total of 240 genes shown in Tables 6, 7, and 8 below. A total of 240 genes shown in Tables 6, 7, and 8 are genes related to liver cancer among the genes contained in the rat genome, and the expression level fluctuates with the formation of dystrophic cancer. It contains genes and genes involved in drug metabolism. In the table, the ACC (Accession) Number and the symbol (common name) of each gene are described. The sequence of each gene Can be obtained from, for example, http: 〃www.ncbi.nlm.nih.gov

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0 £ Confirm OO df / e:) <<! 0Z0 丽 OAV The target gene for detecting the expression level is more preferably at least 20 types, more preferably at least 50 types, among the genes shown in Tables 6, 7 and 8 above. In the method for screening for carcinogenicity of a chemical substance of the present invention, for example, when the expression level of a target gene is detected using a microarray, simultaneous analysis of 20 or more types of genes is performed in consideration of economy and the like. Is preferable. In addition, in order to maintain the reliability (randomness) of the clustering analysis described later, it is desirable to analyze the expression levels of 50 or more types of genes.

More preferably, the method of screening for carcinogenicity of the chemical substance of the present invention detects the expression level of at least one gene among a total of 125 genes shown in Tables 9 and 10 below. Things. The total of 125 types of genes shown in Tables 9 and 10 are the rat gene groups whose expression levels fluctuate specifically in liver cancer among the above-mentioned genes associated with liver cancer. That is, the expression levels of these genes fluctuate in liver cancer cells as compared to normal hepatocytes, and the expression levels fluctuate when a known carcinogen is administered. By quantitatively measuring the expression levels of these genes for a chemical substance of unknown carcinogenicity, the degree of carcinogenicity of the chemical substance can be predicted more efficiently.

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OS CT0 / l700Zdf / X3d 0Z0 Simple SOOZ OAV The target gene for detecting the expression level is more preferably 20 or more, and more preferably 50 or more, out of a total of 125 types of genes shown in Tables 9 and 10 above. Furthermore, it is most preferable to detect the expression level of each of the 125 genes, since the clustering analysis described later can be performed more reliably.

 In the method of screening for carcinogenicity of a chemical substance of the present invention, the expression level of other genes related to the above-mentioned liver cancer may be detected. At this time, it is desirable to include at least 20 kinds of the above-mentioned genes related to liver cancer, and to set the upper limit of the genes other than these genes to 6.0 kinds.

 As described above, the case where the gene related to liver cancer in the rat genome is used as the target gene has been described in detail, but the present invention is not limited to this. There is considerable homology in the genome between mammals, and it is easy to identify other mammalian genes having homology with the gene from a specific rat gene. Therefore, the target gene related to the present invention can be applied not only to the gene related to liver cancer but also to the gene related to liver cancer among other mammalian animal genomes. As mentioned above, humans and mice, which have relatively advanced accumulation of genetic data among mammals, are more suitable than rats.

 Hereinafter, a case where the expression level of the target gene is detected using a microarray will be described. Use of a microarray has the effect that the expression levels of multiple genes can be analyzed simultaneously.

 As the microarray, for example, a two-dimensional microarray in which probes are immobilized on a slide glass (hereinafter, a two-dimensional microarray) may be used, but a three-dimensional microarray is preferably used. The three-dimensional microarray is configured by two-dimensionally arranging a plurality of smiley liquid storage units capable of storing a three-dimensional liquid. Hereinafter, more preferable conditions for analyzing the expression level of the target gene using the three-dimensional microarray will be described. .

When analyzing the expression level of a target gene using a three-dimensional microarray, it is preferable to include the following steps. That is, (1) a step of preparing a nucleic acid labeled with 铸 from mRNA extracted from hepatocytes, and (2) an mRNA transcribed from a target gene. A step of bringing the labeled nucleic acid and the probe into contact with each other in a three-dimensional microarray having a probe that hybridizes with an NA base sequence or the mRNA trap sequence; (3) the labeled nucleic acid and the Detecting a signal intensity that changes based on the binding strength to the probe.

 In the step (1) of preparing a nucleic acid labeled with mRNA as type III, the nucleic acid means any of DNA, RNA, and DNA or RNA containing artificial nucleotides. From the viewpoint of stability and the like, cDNA is preferred. As the labeling method, it is possible to use an RI label, a fluorescent dye label, a label with fluorescent semiconductor particles or metal colloid particles, a biotin label, or the like. Fluorescent labels are preferred because they are the most common, convenient, and safe. Examples of fluorescent substances include Cy3-dUTP, Cy5-dUTP, Fluorescein-12-dUTP, and Fluorescein-12-UTP. These nucleic acids are involved in reverse transcription from mRNA to cDNA. As a result, a fluorescently labeled nucleic acid can be obtained.

 Then, in a three-dimensional microarray having a probe that hybridizes with a base sequence of mRNA transcribed from a target gene or a sequence of the mRNA, a step of contacting the labeled nucleic acid with the probe (2) In the above, it is preferable to use a cDNA or an oligonucleotide as the probe, since it enables detection specific to a base sequence. Hereinafter, the oligo DNA probe is referred to as a probe. The probe preferably has a base length of 20 to 7 Omer, and more preferably 40 to 60 Mer. When the base length is less than 20 mer, non-specific hybrid formation tends to increase, and when it exceeds 7 Omer, it is not preferable in terms of time and cost required for probe synthesis.

 More preferably, the three-dimensional microarray is made of a material having less autofluorescence. An example of a material suitable for the liquid storage portion is aluminum oxide. Here, the liquid storage unit is a minimum unit for immobilizing a probe on a solid phase, and is also called a probe spot.

The three-dimensional microarray can immobilize a larger amount of probes than the two-dimensional microarray described above. In addition, conventional two-dimensional microarrays Because it is difficult to immobilize lobes quantitatively, the amount of immobilization of each probe often fluctuated. On the other hand, the three-dimensional microarray is suitable for quantitative experiments such as analysis of the expression level, because it can quantitatively immobilize probes. When the labeled nucleic acid is brought into contact with the probe, it is desirable to carry out, for example, as follows. For example, a liquid sample is prepared by dissolving the above-described cDNA prepared in the form of mRNA in type III in, for example, sterile distilled water. At this time, as will be described later, a salt or the like can be appropriately added for the purpose of facilitating hybridization with the probe.

 The liquid sample is caused to flow inside and outside the three-dimensional microarray to form a hybrid between the target and the probe. Here, the target is a nucleic acid contained in a liquid sample, and the hybrid is a double strand formed by a nucleic acid having a complementary base sequence. By flowing the liquid sample into and out of the three-dimensional microarray, the liquid sample containing the target can be easily flowed into the space of each liquid storage unit, so that the target molecules and the probe molecules can be reliably contacted, and the hybrid The formation can be performed in a short time.

 When forming a hybrid between the target and the probe, it is preferable to carry out, for example, in an SSPE solution having a salt concentration of 0.1 to 6 times. That is, the SSPE solution is added to the liquid sample so that the salt concentration falls within the above range, and the hybridization reaction is performed. Here, the SSPE solution can be prepared, for example, by diluting an SSPE solution (20-fold). The composition of the SSPE solution (20-fold) is 3.0M sodium chloride, 0.2M sodium phosphate, 0.02M EDTA, pH 7.4. The salt concentration of the S SPE solution indicates the degree of dilution. For example, the S SPE solution having a salt concentration of 6 times is obtained by diluting the S SPE solution (20 times) to 6/20 times. Regarding the salt concentration of the S SPE solution, a higher salt concentration contributes to an increase in signal intensity due to easy formation of a hybrid, but when the salt concentration exceeds 6 times, the rate of increase tends to decrease. On the other hand, when the salt concentration is low, the specificity of the formation of the hybrid increases, but when the salt concentration is less than 0.1 times, the reaction time needs to be lengthened.

When the liquid sample is brought into contact with the probe, the liquid sample is intermittently It is preferable to make it flow 0 times. If the number is less than 30, the frequency of contact between the labeled nucleic acid molecule and the probe molecule is insufficient, and the hybrid formation reaction is often not performed sufficiently. On the other hand, if the reaction time exceeds 200 times, the efficiency of hybrid formation tends to decrease, although the reaction time becomes longer.

 Furthermore, before contacting the liquid sample with the probe, heat the liquid sample in advance at 60 to 100 ° C for 1 to 10 minutes, more preferably at 70 to 95 ° C for 2 to 5 minutes. Is preferred. Further, it is more preferable to perform rapid cooling after heating the liquid sample. By heating the liquid sample, the entanglement (intramolecular bond and / or intermolecular bond) of the target molecule contained therein is released, and the entanglement is prevented from being regenerated by rapid cooling. 'By using the liquid sample containing the target thus pretreated for hybridization, the formation of nonspecific hybrids is suppressed.

 Further, a preferable condition for forming the hybrid is preferably a predetermined temperature range, and the temperature condition will be described in detail later.

 In the step (3) of detecting the signinole intensity that changes based on the binding strength between the labeled nucleic acid and the probe, (3) after the step of bringing the liquid sample and the probe into contact with each other, more preferably Washing is performed by flowing a 33 3 solution of 1 to 6 times the salt concentration inside and outside the microarray. This is intended to dissociate non-specific hybrids, but if the salt concentration of the SSPE solution is less than 0.1 fold, it may also dissociate specific hybridization, and if it exceeds 6 fold, The possibility of forming a nonspecific hybrid between the unreacted labeled nucleic acid contained in the liquid sample and the probe is increased.

 In this way, after the hybridization reaction is completed, the non-specific hybrid and unreacted liquid sample are removed by flowing the SSPE solution with 0.1 to 6 times the salt concentration inside and outside the microarray, and more accurate measurement is performed. It is desirable because it can do. .

Further, the temperature condition for detecting the hybridization reaction and the signal intensity is preferably 30 ° C. to 70 ° C., and more preferably 37 ° C. to 60 ° C. From a hybrid formation reaction point of view, if the reaction temperature is below 30 ° C, non-specific Hybridization tends to increase. In addition, it is preferable to set the reaction temperature to a high value because the specificity of the hybridization reaction is increased.However, if the reaction temperature is too high, the time required for hybridization is increased, or a specific hybrid formation is performed. There is a possibility that it will be hindered.

 In addition, from the viewpoint of signal intensity detection, when the temperature is lower than 30 ° C., it is difficult to dissociate nonspecific hybrid formation. If the temperature exceeds 70 ° C., not only non-specific hybrid formation but also specific hybrid formation may be dissociated, and the overall hybridization intensity tends to decrease. Then, the overall signal intensity is weakened, making it difficult to detect the signal. Here, the term 'signal' refers to fluorescence, radioactivity, chemiluminescence, etc., emitted from a label attached to a nucleic acid molecule hybridized to a probe molecule, or scattered light when a dielectric particle is used as a label. Yes, of these, fluorescence is preferred because of its cost and ease of handling. Preferably, the signal can be measured quantitatively.

 'Finally, clustering analysis is performed based on the detection result of the expression level of the target gene. For example, Steen Knudsen's "A Biologist's Guide to Analysis of DNA Microarray Data" (translated by Satoshi Shiojima, Osamu Matsumoto, Gozo Tsujimoto, "Understanding! Useful! Introduction to DNA Microarray Data Analysis") The analysis can be carried out by the method described in Yodosha, 2002, for example, using analysis software such as Gene Spring manufactured by Silicon Genetics.

 The clustering analysis method includes a clustering process and a process of displaying the results of clustering. If the clustering can be performed to the extent required by the former process, the process moves to the latter process. On the other hand, if the clustering cannot be performed to the required degree in one clustering, the same clustering process is repeated again to perform clustering.

 To perform the clustering process, a hierarchical clustering method and a non-hierarchical clustering method can be used.

Hierarchical clustering is a method of calculating the distance between genes to be analyzed and systematically grouping them hierarchically. In this method, N genes are required. Each gene is plotted as a single point in the N-dimensional long space, and the distance between the two genes is calculated as the Euclidean distance between each data (the distance in each dimension is 2). Square root of the sum of the powers). In this method, the distance between genes is calculated by creating a distance matrix for all genes. Then, it is determined that it is rational to group the genes closest to each other in the space, and a cluster consisting of a combination of genes is formed. By expanding the distance range, when the distance between the already formed cluster and a certain gene falls within the range, the “certain gene” is added to the cluster. To calculate the distance between the gene and the cluster, you may calculate the distance to the closest one in the cluster, but you can use the 'point in the middle of the members in the cluster (cent mouth). : The center of gravity of the cluster, which is the same as the UPGMA or the average linkage method). The distance is extended to include all genes to be analyzed, and clustering is completed. Finally, the clusters are connected to complete the hierarchical clustering method. These clustering steps can utilize computer algorithms. Hierarchical clustering is effective when the number of genes is not large, but its effectiveness can be increased by excluding genes that show no significant difference from the analysis.

The other non-hierarchical clustering method is suitable when the number of target genes is very large, for example, when the heredity index exceeds several thousand. This method attempts to divide the gene population into several clusters. Specifically, the analyst determines the number of divisions m. The computer randomly assigns all genes to each cluster. Calculate the distance between each assigned gene and the center of each cluster (centroid). If a gene is closer to the center of another cluster than the center of the currently assigned cluster, the other cluster One ^-change the assignment. After all genes have been assigned to the closest cluster, recalculate the centroid for each cluster. By repeating these operations several times, the centroid of the cluster does not fluctuate and the algorithm stops. In this method, only the number of clusters requested by the analyst is obtained, and the relationship between the clusters is not shown. Therefore, compared to the clustering by the above division, Search for new clustering directions where rasterization is improved and redo the split. It is determined whether the degree of this improvement is sufficiently small, and if it can be judged that it is sufficiently small, the improvement of cluster integration is stopped. Specific examples of the non-hierarchical cluster method include the K-means clustering method and the self-organizing map.

 In the K-means clustering method, the analyst determines the number of classifications k, and then determines k appropriate points (classification centers) in the attribute space. Each gene is assigned to the closest classification center, and tentatively forms k clusters. Next, the center of gravity of each cluster is obtained, and this is updated to a new classification center. Find the difference between the classification center before updating and the classification center after updating. If the deviation is sufficiently small, terminate the operation. If the deviation is not sufficient, assign all the genes to the nearest classification center using the updated classification center.

 The self-organizing map is different from the K-means clustering method in that centroids move in a two-dimensional grid rather than freely moving in a multidimensional space. The algorithm organizes itself to best fit the data within this grid.

 The distance between each gene is measured in the above method, but the distance can be measured using the leaked distance or other distance coefficients (cosine coefficient, Pearson coefficient), and if necessary, Perform standardization.

 After the above-described cluster unification process, the result of the clustering is displayed, for example, in a linear diagram.

That is, by the cluster analysis described above, the change in gene expression for each individual due to the chemical substance treatment is patterned for each chemical substance treatment group for each of the target genes described above, and finally, the chemical Systematic classification of substances is possible. The phylogenetic classification of the chemical substances obtained in this way is such that strong and weak carcinogenic substances are at both ends, and other chemical substances are arranged according to the carcinogenic strength. .

Similarly, a chemical substance of unknown carcinogenicity is administered to a mammal or caused to act on hepatocytes, and the expression level of the above-described target gene in hepatocytes is detected. This detection result is combined with the detection result of the expression level of the same gene for a chemical substance whose carcinogenicity is known. By performing the tolling analysis, it is possible to detect how much the carcinogenicity of the chemical substance whose unknown carcinogenicity is unknown is higher than that of the chemical substance whose carcinogenicity is known. I have.

 Gene Set>

 As described above, the microarray equipped with a probe that hybridizes with the nucleotide sequence of mRNA or a complementary sequence of mRNA, which is transcribed from a gene associated with liver cancer, among the genes included in the mammalian genome, has been described in detail. However, the present invention is not limited to this. The gene set of the present invention comprises a probe that hybridizes with the nucleotide sequence of mRN'A transcribed from a gene associated with liver cancer, or a complementary sequence of the mRNA, and comprises a carcinogenic screening agent of the chemical substance of the present invention. In the method, the expression level of the target gene may be detected using a method other than the microarray. For example, it is possible to detect the expression status of the above-mentioned gene associated with liver cancer by using methods such as quantitative PCR, RT_PCR, ATAC-PCR, Northern plot, and body map. As described above, the probe used in the microarray of the present invention is a probe consisting of at least two DNAs selected from DNAs of SEQ ID NOS: 1 to 126 or DNAs complementary to any of the DNAs even when separated from the microarray. It can be effectively used as a set of DNAs, and furthermore, the length of the DNAs can be appropriately set to l to 10 mer, ll to 20 mer, 21 to 30 mer, 31 to 40 mer, 41 to By adjusting to 50mer and 51-60mer, it can be used for various purposes.

 In order to analyze the expression state of the target gene, various types of substrates can be applied to the substrate on which the probe can be immobilized. In addition to the three-dimensional microarray, for example, a two-dimensional substrate such as a silicon wafer or glass, a microtiter plate, various glass beads and resin beads, various porous substrates, and various gels can be applied. Example

Example 1

4 types of carcinogens and 6 non-carcinogens at each concentration (% by mass) shown below The seeds were mixed with a basal diet (Oriental MF powder diet) and fed to 6-week-old F344 rats for 2 weeks. One rat received one chemical and three chemicals each. As a control, three F 3.44 rats were similarly fed a basal diet containing no chemical substance for 2 weeks. That is, a total of 3 × 11 1 (10 chemical substance administration groups + 1 chemical substance non-administration group) rats were used.

The concentration of each chemical substance was adjusted in consideration of the toxicity of each chemical substance, such as the LD 50 (half lethal dose).

 Carcinogen

 0.0 1% Diethylnitrosamine (DEN)

 0.0 1% 2_amino-3,8-dimethylimidazo [4,5-f] quinoxaline (MelQx 0.05% Phenobarbital (PB)

 0.3% Clof ibrate

 Non-carcinogenic substances

 1% Caffeic acid

 1% Catechol

 0.05% Nobiletin

 0.05% Garcinol

 0.0 5% Zerumbone

 0.0 5% l -acetoxychavicol acetate (ACA)

 After weighing, the mice were sacrificed at necropsy, a part of the liver was cryopreserved at 180 ° C, fixed in formalin, and histopathologically examined. Next, total RNA was extracted from the frozen liver by the guanidine thiosinate method, and treated with DNase (manufactured by CLONETECH) to obtain 30 g of total RNA, oligo dT primer and reverse transcription. CDNA was synthesized using an enzyme (Takara) and labeled with FITC. A solution of this FITC-labeled cDNA in 6 × S SPE 50 i1 was used as a sample solution (liquid sample). The following three-dimensional microarray system (manufactured by Olympus Corporation) was used for the sample solution to detect the gene expression level.

Figure 1 shows a schematic diagram of a three-dimensional microarray system (Olympus Co., Ltd.). FIG.

 The three-dimensional microarray system shown in FIG. 1 includes a reaction vessel 2 containing a three-dimensional DNA microarray, a microscope 3 for observing a hybridization reaction in the reaction vessel 2, and a support for the reaction vessel 2. And a CCD (charge coupled device) power camera 5 for capturing a fluorescent image of the three-dimensional DNA microarray observed by the microscope 3. The stage 4 is connected to a motor 6 for changing the field of view observed by the microscope 3, and the motor 6 is connected to a stage controller 7 for controlling the motor. The reaction vessel 2 is connected to a pump and a pump driver 8 for stirring the nucleic acid sample solution and flowing it into and out of the three-dimensional microarray, and to a heater and a temperature controller 9 for adjusting the temperature of the DNA microarray. The microarray system further includes the microscope 3, the CCD camera 5, the stage controller 7, the temperature controller 9, the computer 10 for controlling all the pump drivers 8, the monitor 11, and the input unit 12.

 FIG. 2 is an enlarged view showing a schematic configuration of a pump for stirring and flowing the three-dimensional DNA microarray and the nucleic acid sample solution contained in the reaction container 2.

 The substrate 24 of the DNA microarray has a porous three-dimensional structure. A syringe biston pump 22 is connected to the reaction vessel 2 through a tube 21 containing the pressure transfer catalyst. When the syringe / viston pump 22 is moved up and down after the sample solution 23 is placed in the reaction vessel 2, the sample solution 23 moves up and down the substrate 24 and agitation occurs, and the efficiency of the hybridization reaction dramatically increases, It can react quickly.

Based on the nucleotide sequences specific to each of the 125 total genes shown in Tables 11 and 12, 125 oligo DNA probes of 6 Omer were designed. In addition to the probes corresponding to these 125 types of genes, GAPDH as an internal control gene (IC) and an oligonucleotide probe corresponding to lamb da DNA as a negative control gene (6 Omer each) ) It was used. Each of these 127 types of oligo DNA probes Each solid was immobilized on the three-dimensional microarray described above. The nucleotide sequences of these 127 oligo DNA probes are shown in the sequence listing. The sequence of the probe corresponding to GAPDH is shown in SEQ ID NO: 126, and the sequence of the probe corresponding to lamb da DNA is shown in SEQ ID NO: 127.

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df / e :) d 0Z0 simple OAV Next, the three-dimensional microarray on which the oligo DNA probe is immobilized is accommodated in the reaction vessel 2 shown in FIG. 1, and the above-described sample solution 23 is placed in the reaction vessel 2 of the three-dimensional microarray, and a syringe piston pump 22 is provided. The liquid was driven 150 times by using to perform hybridization. The reaction temperature at this time was set to 50 by the heater and the temperature controller 9. After completion of the reaction, washing was performed by driving the liquid once each using the syringe piston pump 22 while updating 50 μl of the 6 XS SPE solution three times. After the washing was completed, a fluorescence image was obtained using a CCD camera 5 mounted on a fluorescence microscope 3 and a WIBA filter one-stroke set (Olympus Optical Industrial Co., Ltd.).

 In the group of rats to which carcinogenic chemicals such as Phenobarbital (PB) were administered, the signal of the probe spot corresponding to genes such as CYP 3 Al, CYP 2B1, and GST Ya, whose expression is expected to increase in liver cancer cells It was clear that the intensity was clearly increased compared to the rats that received the non-carcinogen and the group that did not receive the chemical.

 The signal intensities of the individual probe spots obtained after hybridization 3 were numerically plotted by a computer 10, and GAPDH. (IC) was used as a correction spot to determine the expression level ratio with the group without chemical substance administration. Replaced. This numerical value was subjected to a hierarchical clustering analysis using GeneSpring from SiciconGenecs. The results are shown in Figure 3.

 In Fig. 3, the genealogy shown at the top shows the genes subjected to the clustering analysis described above, classified according to the similarity of the change in expression. According to this gene line, the gene expression ratio of each chemical substance administration group was patterned (omitted in FIG. 3), and based on that pattern, the above was described! The system diagram on the left classifies 0 chemical substances. Specifically, it was found that the ACA side was a chemical substance having less carcinogenicity and the Pheno ob arbita1 side was a chemical substance having more carcinogenicity, with ACA and Phenobartarbital as both ends. Example 2

As in Example 1, 0.05 wt% A uraptene was mixed with the feed and 3 F 3 Forty-four rats were administered for 2 weeks, and the expression level of the target gene in the liver was analyzed using a three-dimensional microarray. Based on the expression level of this gene, clustering analysis was performed together with the expression level of the same gene in a group administered with a chemical substance having a known carcinogenicity using Gene Spring of Silicon Genetics. Figure 4 shows the results.

 As shown in FIG. 4, Auraptene was classified between Zerubumbone and CaffeinecAcid, and was confirmed to be a non-carcinogenic substance. As a result, administration of a compound to rats for a short period of two weeks made it possible to predict the carcinogenicity of the chemical from changes in gene expression in the liver. INDUSTRIAL APPLICABILITY The method for screening for carcinogenicity of a chemical substance of the present invention detects the expression level of a gene associated with hepatocellular carcinoma among genes included in the genome of mammals. In this way, a carcinogenic screening of a chemical substance can be performed in a shorter time and at lower cost than in a method of administering a chemical substance to an animal and detecting the cancer pathologically. More specifically, the carcinogenicity is determined by performing a clustering analysis on the basis of the results of detection of the expression levels of the genes by a chemical substance of unknown carcinogenicity and a chemical substance of known carcinogenicity. It becomes possible to know how much the unknown chemical substance has carcinogenicity as compared with the chemical substance whose carcinogenicity is known.

 Further, the microarray of the present invention has a probe mounted with a probe that hybridizes with a nucleotide sequence of mRNA transcribed from a gene associated with liver cancer or a complementary sequence of the mRNA, among the genes included in the mammalian genome. Therefore, it can be suitably used for carcinogenicity screening of chemical substances. At this time, if a three-dimensional microarray is used, reproducibility of gene expression level analysis is good and efficient. In addition, the gene set comprising the above-mentioned probe can be used to apply to the carcinogenic screening of chemical substances by various methods other than the microarray.

The method of screening for carcinogenicity of a chemical substance of the present invention provides a method of screening a chemical substance of unknown carcinogenicity, as compared with a chemical substance of known carcinogenicity. Can be evaluated. Further, the microarray and the gene set of the present invention can be suitably used for screening the carcinogenicity of a chemical substance.

Sequence listing

 SEQUENCE LISTING

110> Olympus Co., Ltd.

 <120> Screening Method for Determining Carcinogenesis of Chemical Compound <130> 04P01861

<160> 127

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acatgaggat gatccagcta cacaatgggg agtacagcaa cgggaagcac ggctttacca 60 <210> 27

<211> 60

<212> DNA

213> Rattus norvegicus

<400> 27

aacctccatg gtctgtttgg gcggaagaca ggccaggctg ctggattctc ttacacagat 60 ku 210> 28

<211> 60

 <212> DNA.

<213> Rattus norvegicus

 <400> 28

gcagttatcc atgagattca taggttttca gatcttgtcc ctattggagt accacacaga 60 <210> 29

<211> 60

<212> DNA

 <213> Rattus norvegicus

 <400> 29 '

caagtaccac tctgaggtat ggactcctgc tgctgctgaa acacgtggat gtcacagcta 60 <210> 30

<211> 60

<212> DNA

 <213> Rattus norvegicus

C 400> 30

ctacgcttgc tcttgtggtt tcggtgactt gcctcagcct cctttcagtg tggacaaaac 60 <210> 31

<211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 31

tttctcagca ggaaaacgga tgtgtgcagg agagggccta gcccgcatgg agttattttt 60 <210> 32

<211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 32

gatactgtga tggaaatgga atacctggat atggtgttga atgaaaccct cagattgtat 60 <210> 33

 <211> 60.

<212> DNA

 <213> Rattus norvegicus

C 400> 33 ttactgttct agaaacactg gggaggtttc gacatgctct cgctatattt tattttactg 60 <210> 34

C 211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 34

agcctcttcc tggcacctgg gaagaatatc tggagaaatt cctcgctgga aatgtggcct 60 <210> 35

 <211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 35

acctcaaggg caaagccaag tgggactcgt ggaacaagct gaaaggaact tccaaggaaa 60 <210> 36

<211> 60

<212> DNA

 <213> Rattus norvegicus

C 400> 36

gagagaggag tcagagagct gttagtacca tgtctacccg ttaccatgga ccgcaatgtg 60 <210> 37

C 211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 37

gaaccgtttc aactctcgta cttactccaa gttcaaaaac tccctcgtgg tctccttcct 60 <210> 38.

<211> 60

<212> DNA

213> Rattus norvegicus <400> 38

cctacctgcc tgtcaatgaa tcctttggct tcactgccga ccttcgatcc aacactggtg 60 <210> 39

<211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 39

cggtacagca agcgccccct ggtggttgta tactacagcg tggacttcag ctttgattac 60 <210> 40

 <211> 60 '

<212> DNA

 <213> Rattus norvegicus

<400> 40

tgccaccatg aacttctccg gcaagtacca agtgcagagc caagagaact ttgagccctt 60 <210> 41

<211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 41

ggtgccatgg ccaaaccaga ctgcatcatt accctcgaga acaacaacct caccgtcaaa 60 <210> 42

<211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 42

accaaaccaa cggtgattat cagtcaggaa ggcggcaaag tggtgatccg gacacaatgc 60 ku 210> 43

<211> 60

<212> DNA

<2s n13> ttuorvelcus,

 g <21> attus norvicus3e ..

g <21 noi3rvecus

¾ srtow »§60 g2 Rasus no13rveicus <212> DNA

213> Rattus norvegicus

<400> 48

acatctaggg tgggggagtt ctggagggag aagcaggcag ataaatcaga gtgggggttg <210> 49

<211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 49

gaccttctga ttcaagagct actggcctgg atggcggacc aagcagagct ctgtcgactg <210> 50

<211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 50

aaaactcacc atttccatcg tctctgtctc tcggccccct gatctttccg c.atggttgtg <210> 51

<211> 60

K 212> DNA

213> Rattus norvegicus

 <400> 51

tcggaaaaga ggtagggcag aggctcccac ggctgatatc tggaacttcg tcggctctaa <210> 52

 <211> 60

 K 212> DNA

 <213> Rattus norvegicus. <400> 52

caagtgctgt agtgggtccc tggtggaaag acggccatgt ttctctgctc tgacagttga ku 210〉 53 <211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 53

cagcagcctc cccaatgtga agaagttcct gcagcctggc agtcagagaa agcttcccat <210> 54

<211> 60

<212> DNA

213> Rattus norvegicus

<400> 54

cttctccagc tttgtcagcc ccatctcctc aacctcaccc cagtcatgcc cacatagtct <210> 55

<211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 55

gcagctttga gtccacacct ctgtctacgc agcagctatg ccgccgtaca ccattgtgta <210> 56

C 211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 56

agtcaactac cccctttcct caaactctca tatacccgat ggacaggtct ctggaacatt <210> 57

<211> 60

 <212> DNA.

<213> Rattus norvegicus

<400> 57

ccctcataac tgtccgccca cacccaatgg ctttctgaga gaagcaccac tctggttagc <210> 58

<211> 60

<212> DNA

 <213> Rattus norvegicus

Shelf> 58

accgcagaaa atatgcccgg cgtcccgtct aaggagacca atccatacaa ccattccccg 60 <210> 59

<211> 60

 <212> DNA ''

<213> Rattus norvegicus

<400> 59

gagcttgctg tgagaacgtg atcggatata tgcccatccc tgttggagtg gcaggacctc 60 <210> 60

<211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 60

tgcagagcga tttgttgttg aggcaggaaa gctcttgggt ggactggaca tgctcattct 60 ku 210〉 61

<211> 60

212> DNA

 <213> Rattus norvegicus

<400> 61

ctgaatcagg ccgctctgta ccgcctcagt ggagactcga atcctttaca cattgacccg 60 ku 210〉 62

 <211> 60-K 212> DNA

213〉 Rattus norvegicus

<400> 62 gtcttctctc gcttctcact ttaggaggcc caaaacccac tccaaccgca tctaccgcat <210> 63

C 211> 60

<212> DNA

 <213> Rattus norvegicus

C 400> 63

attatatctt ggacctgcag atcgccctgg actcgcaccc cactatcgtc agcctgcacc ku 210〉 64

C 211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 64

aacgagcaca agtcagtctg aggtaggggc ctttcagtgg gttcagggag gaaggttagc <210> 65

<211> 60

K 212> DNA

 <213> Rattus norvegicus

C 400> 65

gaaaaccaaa cccggcaggt ggtgtgtgac cttgggaacc cgaagaaggc tggaactcaa <210> 66

 C 211> 60

 <212> DNA

 <213> Rattus norvegicus

 C 400> 66

 tgagcacggt tcccagtttg ccatgcgttt tgatgactga tagaccaaag tcttattcct <210> 67

 <211> 60

 <212> DNA

<213> Rattus norvegicus

gi1 rt norvec23> atus <

TOis norvec 13> ttu ..

 ggggggg ggggTOgg caccttcccaaacat acat: ccttccatccaat acaaaaa σϋ actat ... g norveicus213> ttus <..

g noricusttusve .. ggggggggggTOcca caacaccta ctc cacrtcaacc acattctatatactttccccadccc TOO ...., ....

60 TO <213>? Aftlrlls novecus.

ggggTOggg g aatcacggtctc acaata acccaaaacc ctaccac ataccaaacaacaccc 60.

g 21 R3>asu nrvisoecus

g 21 R3> asu nrvisoecus

gggggggggg gggtccttccgg cttaattctcactctttacattcatcactc atetacl 60 ......-........ g <213 nrvittusoecus ..

TOgMg ggggg ¾¾OT ctacagatt aataccacc ctccaaccattccrfowOrf caicaatc 60 ..... g 13V nrvioecus K 212> DNA

 <213> Rattus norvegicus

<400> 77

gagtggcggg gaggagtgct tccttatcag tcagttttgt tgtgaggtca ggaaggacga 60 <210> 78

C 211> 60

<212> DNA

213> Rattus norvegicus

Ku shelf> 78 '

ttcttgtaag gacgaagcgg gactggcttg tgaaacaaag aggctgggat gggtttgtgg 60 <210> 79

<211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 79

caccaccacc acagttgcag aaaagtacaa acaccgaggg gagggagagc gcaaagacat 60 <210> 80

<211> 60

212> DNA

213> Rattus norvegicus

<400> 80

aaatatcgag atgtcagatc tgtccttcag caaggactgg tctttctaca tcctggctca 60 <210> 81

<211> 60

<212> DNA

 <213> Rattus norvegicus ·

<400> 81

aagccatcct tcgagtcttc cagcccaaag ccgtctggct gaaattgagt aaccgtttga 60 <210> 82 C 211> 60

<212> DNA

 <213> Rattus norvegicus

C 400> 82

tatcggcctc ctgtactccc tgagcggacc ggatctctct acagccctca ttcacttcag ku 210〉 83

C 211> 60

K 212> DNA

 <213> Rattus norvegicus'

<Shelf> 83

accgctgttc ttcgagtctt gctgcacgcc ccgttctgtg tttatccacc cgtaatgatg <210> 84

C 211> 60

212> DNA

213〉 Rattus norvegicus

C 400> 84

tcgcagtccc cacctggtgt tctttctagc catctacagt acagtcatgc actcaaatta <210> 85

C 211> 60

K 212> DNA

 <213> Rattus norvegicus

C 400> 85

tggagagacc aaccccgcag actctaagcc tgggaccata cgaggagact tttgcattca nice 210> 86

C 211> 60

 <212> DNA-<213> Rattus norvegicus

 C 400> 86

attgttctat tgtggactac tgaatctctc tcctctcccc cgtcccctat tctcccttcc <210> 87

<211> 60

<212> DNA

 <213> Rattus norvegicus

C 400> 87

aggagagcgg gatggaccgt caccctgcag cctgtgcttc tgctagtatc aatgtataga <210> 88

C 211> 60

 <212> DNA ''

<213> Rattus norvegicus

<400> 88

gtcctcaccc aagtcagaaa ggcttaacat tcctacctcc aaggcaaaca tcccaaatca <210> 89

<211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 89

ggactgtccc catgcccact ctacccccac tcctgtgcac ctcgattcta ttttccacaa <210> 90

<211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 90

gacatgtctc catgtcgagg tacttgttcc accacgcaga cctccctgag accccttcct <210> 91

 <211> 60. <212> DNA

 <213> Rattus norvegicus

<400> 91 atgtcagaag aggacttcga gaaagcattc aacgccaggt tcccggggtg catgaaaggt 60 <210> 92

<211> 60

<212> DNA

213> Rattus norvegicus

<400> 92

atgctgtggt gatctcctgt gcaa & ggacg gggtgaagtt ttctgcgagt ggggagcttg 60 <210> 93

<211> 60

<212> DNA

213> Rattus norvegicus

<400> 93

cgtggggaaa actgcttgaa ggagcggagg agccgattgt gtactcggat gatgaagaac 60 ku 210> 94

<211> 60

K 212> DNA

 <213> Rattus norvegicus

<400> 94

agcctgatct ttccccaaca ctccacagcc ttggatccgc ccactttcac ttttcccttg 60 <210> 95

<211> 60

<212> DNA

213> Rattus norvegicus

<400> 95

aagacggcac tagcctccat ccagcctcca tctttgtgta cacctgcatg caagaagacc 60 <210> 96.

<211> 60

<212> DNA

<213> Rattus norvegicus

g ttaacactt

 ggOT cacttacacc aaa atcacarfct 60 ..

ggggg atccac accaaaccctatcttc accaccccac caaaaccaca acataatt 60 ..

 g 21> 3attus norveicus.

TOgggggsTOTOgTOTOgggacccac attaaac rtrtrtrfrfsaacttt caatcctcc tttct .......

 g Rs norviattuecus

ggggg ggTOggggggggaatatcattcatacacacctctttacaaa ataac 6act0 ......- <213> Rattus norvegicus

<400> 101

agcaagcaga tgtggtagtg gcggaagtga cacagccatc cttgggtgtt ggctatgaac <210> 102

<211> 60

<212> DNA

Gu 213> Rattus norvegicus

<400> 102

gcggcgctac gccaggaccg a'tgggaaggt tttccagttt cttaatgcca aatgtgagtc <210> 103

<211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 103

ggcgtgacaa agggcaaagg ctacaaaggg gtgaccagtc gttggcatac aaagaagctg <210> 104

C 211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 104

ccctctacag aatgggtttt cccgaagctg ccagctcctt cagaacgcac cagatttcag <210> 105

<211> 60

K 212> DNA

 213> Rattus norvegicus

 <400> 105-ggagaacgca aaccgtccag cgcagcttac cagaaggcac ccactaagga gttttatgca <210> 106

<211> 60 <212> DNA

 <213> Rattus norvegicus

C 400> 106

gaggccgtcc ccatgtctct gccccctcaa gtgaagttcg accacccttt cattttcatg 60 <210> 107

<211> 60

<212> DNA

213> Rattus norvegicus

<400> 107 '

catgacacca cacttactgg ctacctcctc cctgatgccc ttctctggct ttctgatggg 60 <210> 108

<211> 60

K 212> DNA

 <213> Rattus norvegicus

C 400> 108

tcaatcactg agggctttct gttctgttga ccttgtgtct cacatgctca cggatggggc 60 <210> 109

<211> 60

K 212> DNA

 <213> Rattus norvegicus

Shelf> 109

cacagatgtg attggaaacc ctgaggagga gagacgagct gctttctacc accagccctg 60 ku 210〉 110

C 211> 60

<212> DNA

 <213> Rat tus norvegicus.

Shelf> 110

aggcaagcgg tgaaccagtt gtggtgtcag gacagattac aggattaact gaaggcgagc 60 <210> 111 gggTOggTOggOTg ggggggTOg aattat atac cacacaatlcaaa attatc 60-.. gg gggTOggggcatccaaataaac aaacc06.

gTOggtcctac:a aCaatca cctttctt 60..,...

gTOggtcctac: a aCaatca cctttctt 60 .., ...

ggggTOgggtaccaaa actcaatcaatcatt 60 ...-- gg ccccaccacctct cccccttccatta06 ....-- g <2 n13 Ratusorveiscu .. <210> 116

C 211> 60

<212> DNA

 <213> Rattus norvegicus

C 400> 116

gaagctgaga ctaagttgag gcaaatctgg ttggacatcg tcctgccctg acataagaca <210> 117

<211> 60

212> DNA

 <213> Rattus norvegicus

<400> 117

agtgtgggtt ggttagggga aggcaaactg gtgagaacat ggatttctgt ggtgcaggca <210> 118

<211> 60

<212> DNA

 <213> Rattus norvegicus

<400> 118

gccacccatt atcgccttgg ttcgcccatc agagttgtaa gaaaatggca gacaagccgg c 210> 119

<211> 60

K 212> DNA

 <213> Rattus norvegicus

C 400> 119

ggctccctct catcagttcc atggcccaga ccctcacact cagatcatct tctcaaaact <210> 120

K 211> 60-K 212〉 DNA

 <213> Rattus norvegicus

gTOgggggcccaaat tttcctca acacaatc σϋ.. <400> 120 g1 w norvi <23> ¾rtcwecus g gggTOgggccttccccact taaaaaa ctta 60 ..

gTOgggggcccaaat tttcctca acacaatc σϋ ..

gg aaaaata

3oBB¾...ooS

3oBB¾ ... ooS

 LZ \ c 00 丽 BpqniBq c ε> ma <ζιζ>

09 <UZ> LZl <Q \ Z> SSBOBBOBB ^. BOOOO SEOO o .BOo¾..BBO BOSBS ^ OBOB BOOOOOSBO ^.

 9ZI <00f> sno SaAJou snq.q.sji g | g

 VNO <ZIZ> 09 πζ> 9Ζΐ <Q \ Z> B ^ SISBBO ^ O Boo3..q.oS ^ BSOOBSSBBS q.S3BSoooo. 05.035.050 ^ 5. SBOOBSSBBO

 SCT Ku 00〉

S9

 0Z0 丽 SOOZ OAV

Claims

The scope of the claims 1. A carcinogenic screening method for a chemical substance, comprising detecting the expression level of a gene associated with liver cancer among the genes included in the mammalian genome. 2. Administration of a chemical substance with a known carcinogenicity or a chemical substance with an unknown carcinogenicity to mammals or the action on their hepatocytes, which is related to liver cancer in hepatocytes of the mammals in the chemical substance treated group and the untreated group The chemical expression according to claim 1, wherein the cancer expression is detected by detecting the expression level of the gene and performing clustering analysis based on the detection result, thereby screening the carcinogenicity of the unknown chemical substance. Carcinogenic screening method for substances. 3. The carcinogenic screening method for a chemical substance according to claim 1 or 2, wherein the mammal is any one of a human, a mouse, and a rat. 4. The method according to claim 1, wherein the expression level of at least one gene is detected among a total of 240 kinds of genes shown in Tables 1, 2, and 3 below. A method for screening for carcinogenicity of a chemical substance according to the above item. 2ΜΡ0 εεειεο Ichigo  1-JS3 6892 m ~ NN mpo εειεο— Ν  0 dJ3 IPUOQ a Ichigo iquoo

 46 OSLSO—WN  10 ー 刚 IBO 0S9S10 "MN  edsso  uiua Ldseo 10  刚 εΓ 刚 2 210—rigid  JTBD 66S 0—ιε is-i / a 89220 ー  ιο—Go Sir OS80S0  666910 "NN 8669L0" WN  nw L222L [~ m  xeg 6S0 0—go  Legd uiiv S S— WX  ZdAO £ 68 l0dV ssv  d ε½ιεο "" Go L6J l LOl l  9P2dAO (on K eodv  6 ^ d ιοςεοΜ 19 t0 〖0— ΪΖ  6S-L LZ10 ~  I L d BF 10 1 Z 2LLSL0 "^ N  8S 2 〖(T Go  69 temporary 8 1720—go od \ / 66 Kazuyoshi Kasumi

 s. — Σβχυν  TS10 IB uy epso TdT 650εΐ0 "ΝΝ 11 d-iQ ■ οκηεο—  dqqaJQ ΐδεεεΓ ^ Ν ε pen stsoHN  09x03 09S6L0 one ring οεζεεο one WN  BSLjV 868210 ~ WN  LO—WN; 6V  IJ lpv ε9οειο ~ Ν  ΠΤ0 οεςο ー 刚 Haru 麵 【0ichigo esu | p3 059ΐεθ ~ Η L6 so-called  qi  e. 80— Ν 117 i¾V L • ON :) V ·. Ν ΐ 挲 99 CT0 / 1-00Zdf / X3d 丽 SOOi OAV ZPI 090E10 "WN 刚 S90LeO" WN B19q-06dSH  ieoiso "WN  iqiLPSH. ■ 0—  ■ 1982L0 "HN IxouiH 08S2L0" ^ N  m 9569 IX Kasumi aichi WN  J061UH ε ιε lo— 刚  £ .9220 "WN O0-9NH  麵 ■ 10—WN 6  9 thigh 0 ichigo SH L0—go III 6 W S IO—WN 8 SSCfWN  9 w 2 back 0 1 so-called ει—go  E Lord SH1S9 SS998n  mzor  Basket 10 Ichigo  i ειο ο Ichigo  dew 88CS0 ~ NN 9-91S9 t759Z9X  S dew 9 0 ー person 1S9 Ζ £ 6ίΟ)  ε “WN 906SS0—Tough ε-Ran WN Wi Ceramic 9280SO-1 LOl 9ειειο ~ ΝΝ JOd-9  haze. 01 18018 Γ 刚  J-ΪΙ 96 20—Go Hog  Jdan 96S210 "WN OBU9 2S 10— 刚  Old. LOl l suTpni9 οε 麵 ー 剛  IJUJB "! 8ei £ lO ~ WN IQfO  2SBJ> |  Lzwzo ~ nn  JP Ζ90εΐ0 ~ ΗΝ z  punp εΓ 硬 l £ l 16 unr ςε8 ο— WN LOiuj-mil 69S610— 刚 90S6L01W  8S8 刚 V  V1NI 66  q Egg I ςςεεςο— 刚 917 Okiichi Go  L0—WN 8 l W  JIJ-BI Hall PPBd  LJ-6I 9 ban ΪΖΙ ΪΖΙ 18U89 # 00V • ON ■ g; ^ •. N s 挲 Zdf / e :) d 0Z0 丽 OAV

 ella  2,9 Z Zdf / ェ :) d 0Z0 丽 SOOZ OAV 5. The expression level of the chemical substance according to claim 4, wherein the expression level of at least 20 kinds of the total 240 kinds of genes shown in Tables 1, 2, and 3 is detected. A carcinogenic screening method. 6. Among the total 240 kinds of genes shown in Tables 1, 2 and 3 above, at least The carcinogenic screening method for chemical substances according to claim 5, wherein the expression levels of 50 types of genes are detected. 7. The expression level according to any one of claims 1 to 3, wherein the expression level of at least one gene is detected among a total of 125 types of genes shown in Tables 4 and 5 below. Carcinogenic screening method for chemical substances. Table 4  ACC # Gene No. ACC # Gene N .012488 A2m 41 NM-I 145878 Fapb5 U28152 of2u-globulin NM— 030832 Fabp7 NM— 031760 Abcb11 X05834 FBN NM-1 012891 Acadvl NM— 012559 Fgg M J 34432 Agt AF014828 Figf NM-1 012898 Ahsg NM— 012792 Fmo1 NM— 031010 Alox12 NM.024127 Gadd45a NM— 012738 Apoal one 017251 Gjb1 NM— 013112 Apoa2 N _019630 Gludins NM— 012737 Apoa4 NM— 012571 Got1 N .012501 '. Apoc3 51 NM— 013177 Got2 NM J 38828 Apoe N _030826 Gpxl NM— 017134 Arg1 MM— 017013 Gsta2 M36708 Ass NM— 177426 Gstm2  One 030850 Bhmt N _138974 Gstp2 NM— 022399 Calr NM 053448 Hdac3 — 012727 Camk4 NM— 022179 Hk3 N _012520 Cat M33648 H G-CoA NM_053021 Clu N .013134 Hmgcr NM— 017202 Cox4a N _017080 Hsd11b1 053586 Cox5b 61 NM—024392 Hsd17b4 MM— 019360 Cox6c S45392 HSP90- beta N _017096 Crp NM_013060 Id2 U22893 Csda ー 013144 Igfbpl MM one 031315 Cte1 S58528 thigh  NM— 013156 Ctsl NM_031768 Itgae N .012839 Cycs N _019369 Itih4 Μ1Ϊ251 Cyp2bl NM_017138 Lamrl NM_019184 Cyp2c11 1 022195 Lif  One 031839 Cyp2c23 NM— 013136 Mak K03501 Cyp2c9 71 NM_031643 Map2k1 M10161 Cyp3a1 N _133283 Map2k2 NM— 012942 Cyp7a1 NM— 017246 Map2k5 NM-I 022513 D / T-ST N _053887 Map3k1 NM— 031853 Dbi NM.053842 apkl NM J 38877 Dial N _017347.Mapk3 1 053354 Dnmtl NM— 031622 Mapk6 NM— 017245 Eef2 NM 021846 Mcl1 ー 053849 E “p70 NM— 022673 Mecp2 NM— 012556 Fabpl X16956 G 9 SJJ_U 丄  ： ^ 丄 剛 剛 oiqsmi 6 upper Ichigo  10-  18 Simple — 'ZldS t ^ S869X ojeds 9 III IPOS 050 10 ~ NN  SPOJEUJS S86tS0 "NN  SB. TS 60 Ikko  Devil 10—  tBUTdJ9S 6LS 0-1 HN  Ζ8θεΐ0 ~ Ν S. . S 80S8SL ~ WN

Toy L0L Ceramic OA0900rV B attached luod  t? P. Td 889080—  865LS0 ~ NN lOiiiJBd 99 ー  BUOd  . d. ε εο »16 8662 lO W 阔 8 SSOlW Ceramic 0Ζ80ε0 ~ ΝΝ

 2SO 89 £ S0 ^_ WN  905ε m iq willow 卿 Lord 0861S0—Tough tSOLS0 "MN  09S0 S S6W 6t¾t¾r 刚 zux 丄 ιεεεςο ~ ΝΝ 121 19--18 9U8Q #oov ON 9U99 # 00VON s 挲 £ T0 / l700Zdf / X3d 0Z0 Simple SOOZ OAV 8. The carcinogenesis of the chemical substance according to claim 7, wherein the expression level of at least 20 genes among the total 125 genes shown in Tables 4 and 5 is detected. Sex screening method. 9. The carcinogenesis of the chemical substance according to claim 8, wherein the expression level of at least 50 kinds of genes among the total of 125 kinds of genes shown in Tables 4 and 5 is detected. Sex screening method. 10. The method of screening for carcinogenicity of a chemical substance according to claim 9, wherein the expression levels of a total of 125 kinds of genes shown in Tables 4 and 5 are detected. 11. A probe comprising a probe that hybridizes with a nucleotide sequence of mRNA transcribed from a gene associated with liver cancer or a complementary sequence of the mRNA among genes included in mammalian genomes. Microarray. 12. The microarray according to claim 11, wherein the mammal is any one of a human, a mouse, and a rat. 1 3. A microgroove array equipped with a plurality of types of probes, wherein each probe is an mRNA base transcribed from at least one of a total of 240 types of genes shown in Table 1, Table 2 and Table 3. The microarray according to claim 11 or 12, wherein the microarray hybridizes with a sequence or a complementary sequence of the rnRNA. 14. A microarray equipped with at least 20 types of probes, each probe having at least 1 of the total 240 types of genes shown in Tables 1, 2 and 3 above. 14. The microarray according to claim 13, wherein the microarray hybridizes with a base sequence of mRNA transcribed from two genes or a complementary sequence of the mRNA. 15. A microarray equipped with at least 50 types of probes. That the probe hybridizes with the nucleotide sequence of mRNA transcribed from at least one gene among at least one of the 240 genes shown in Tables 1, 2, and 3 above, or with the complementary sequence of the mRNA. The microarray according to claim 14, characterized in that: 1 6. A microarray equipped with a plurality of types of probes, wherein each probe has a nucleotide sequence of mRNA transcribed from at least one gene out of a total of 125 types of genes shown in Tables 4 and 5, or 13. The microarray according to claim 11, which hybridizes with a complementary sequence of the mRNA. 1 7. A microarray equipped with at least 20 types of probes, wherein each probe has a nucleotide sequence of mRNA transcribed from at least one of the 125 total genes shown in Tables 4 and 5, or 17. The microarray according to claim 16, wherein the microarray hybridizes with the mRNA trap sequence. 1 8. A microarray equipped with at least 50 types of probes, each probe having mRNA bases transcribed from at least one of the 125 total genes shown in Tables 4 and 5 above. 18. The microarray according to claim 17, wherein the microarray hybridizes with a sequence or a complementary sequence of the mRNA. 1 9. A microarray on which at least 125 types of probes are mounted, wherein each probe is a nucleotide sequence of mRNA transcribed from at least one gene of a total of 125 types of genes shown in Tables 4 and 5 above, or 19. The microarray according to claim 18, which hybridizes with a complementary sequence of mRNA. 20. A gene set comprising a probe mounted on the microarray according to any one of claims 11 to 19. . 21. A method for screening a cancer-causing chemical substance according to any one of claims 1 to 10, wherein the expression level of the gene is detected using a method comprising the following steps: Law.  (1) A step of preparing a nucleic acid labeled as type II with mRNA extracted from hepatocytes;  (2) contacting the labeled nucleic acid with the probe in a three-dimensional microarray having a probe that hybridizes with a base sequence of mRNA transcribed from a target gene or a complementary sequence of the niRNA;  (3) a step of detecting a signal intensity that changes based on a binding intensity between the labeled nucleic acid and the probe. 22. A probe DNA set consisting of at least two of the DNAs of SEQ ID NOS: 1-126 or DNAs complementary to any of them. 23. The probe DNA set according to claim 22, wherein the length of the DNA is 1 to: I Omer. 24. The probe DNA set according to claim 22, wherein the length of the DNA is 1 :! to 20 mer. 25. The probe DNA set according to claim 22, wherein the length of the DNA is 21 to 3 Omer. 26. The probe DNA set according to claim 22, wherein the length of the DNA is 31 to 4 Omer. 27. The probe DNA set according to claim 22, wherein the DNA has a length force of 41 to 50 mer. . 28. The probe DNA set according to claim 22, wherein the length of the DNA is 51 to 60 mer.