KR101062784B1 - Biomarker for Identifying Mirex Specific Exposure and Identification Method Using the Same - Google Patents

Abstract

The present invention relates to a biomarker for identifying whether mirex specific exposure is one of the residual organic pollutants and a method of identifying the same, and specifically, a biomarker in which gene expression is increased or decreased specifically by Mirex. And it relates to a method for identifying the specific exposure to Mirex using the same, the biomarker of the present invention by using the reaction genes selected through the DNA microarray chip as a biomarker to monitor and It can be useful for the determination and can be used as a tool to identify the mechanism of toxic action specifically induced by Mirex.

Description

Specific biomarker for identification of exposure to mirex and the method of identification using the same}

The present invention relates to a biomarker for confirming whether or not a mirex-specific exposure and a method of identifying the same, and more particularly, a biomarker that specifically increases gene expression by Mirex and a mirex-specific method using the same. It relates to how to check the exposure.

Among the persistent organic contaminants, Mirex was used mainly as an insecticide and a flame retardant. Mirex has been reported to indicate skin, liver, nervous, reproductive and endocrine disorders. Mirex was produced commercially and banned due to known risks. However, Mirex is still detected and is detected even where it has not been used.

Mirex is known to have a half-life of about 10 years in soil, adsorbed mainly on low quality and suspended solids, hydrolysis reactions in water and high concentrations of aquatic organisms. In addition, lipid retention in the body is known to be very strong. In a factory in Hopewell, Virginia, mirex has caused 76 of 148 workers to cause serious neurological symptoms. This is known as "Kepone shake," which is characterized by symptoms such as tremors, gait problems, behavioral changes, opsoclonus, arthralgia, headache, chest pain, weight loss, hepatomegaly, paraplegia and erectile dysfunction. (Morgan, DP et al., Bull Environ Contam Toxicol. 22: 238-244 1979). Many of the major toxic symptoms of Mirex have been identified in animal studies, with major target target organs being the central nervous system, liver, adrenal glands and testes. Like other organochlorine insecticides, Mirex is involved in the formation of malignant tumors in liver tumors and organs other than the liver, and females are more sensitive than males. Mirex causes hepatomegaly, especially in males, and as dose increases, the likelihood of tumor nodules and liver cancer increases (Chambers, JE and Trevathan, CA Toxicol Lett. 16: 109-115, 1983).

The concentration and accumulation of persistent organic contaminants in food is well known, and once chemicals enter the body, in vivo metabolism is exceptionally slow because of the partially complex aromatic ring structure and chlorine. This is because of the degree of substitution. This is because such a ring structure is extremely difficult to be removed by enzymes in tissues in vivo. Fat-soluble Mirex, which has a high fat / water partition coefficient, enters body organs with high fat content (liver, kidney, nervous system, and fat cells) to exhibit some physiological effects or remain stored in the case of fat cells (Ministry of Agriculture and Forestry). , Agri-food hazard information manual).

In addition, although some studies on the degree of carcinogenicity and gene expression of persistent organic pollutants have been reported, it is almost limited to the study of dichlorodiphenyl-trichloro-ethane, which is known as a representative persistent organic pollutants. Despite the possibility of Mirex's human carcinogenesis (IARC, Group 2B), there is not enough risk assessment data in humans, and the detection method for exposure is also GC-MS (Gas Chromatography-Mass Spectrometer) or HPLC (High). It is limited to classical methods such as Performance Liquid Chromatography. Quantification is possible using GC-MS or HPLC methods, but appropriate conditions for analysis are required and expensive equipment is required. Therefore, faster and easier screening methods, such as real-time reverse transcript polymerase chain reaction (PCR) or DNA microarray chips using primers, can be used in humans through rapid risk assessment. It is an important task to discover and utilize molecular indicators that can detect the toxic effects of genes and the expression of genes, and to take appropriate measures and control against the exposure of Mirex.

Several genome sequencing projects, including six mammals and 292 microbes, have been completed and reported to the National Center for Biotechnology Information (NCBI). Genome-wide expression studies are being conducted to study the function of genes based on the vast amount of data obtained. DNA microarray analysis is performed to analyze the expression of thousands of genes in one experiment (Schena, M., et al. , Proc. Natl. Acad. Sci. USA 93: 10614-10619, 1996). .

Microarrays integrate a set of oligonucleotides of cDNA (complementary DNA) or 20-25 base pairs in length on glass. cDNA microarrays are produced by labs in schools or by companies such as Agilent, Genomic Solutions, or the like, by mechanically immobilizing cDNA collection on a chip or by using ink jetting (Sellheyer K. et. al., J. Am). . Acad Dermatol 51:.. 681-692 , 2004). Oligonucleotide microarrays are made by Affymetrix's method of direct synthesis on a chip using photolithography, and Agillent's, etc., are produced by immobilizing synthesized oligonucleotides (Sellheyer, K.). et. al., J. Am. Acad. Dermatol . 51: 681-692, 2004).

To analyze gene expression, RNA is obtained from a sample such as tissue and hybridized with oligonucleotides in a DNA microarray. The resulting RNA is labeled with fluorescence or isotope and converted to cDNA. Oligo microarrays are mainly labeled with two different fluorescences (eg, Cye3 and Cye5) to perform hybridization reactions simultaneously on the same chip by labeling the control and experimental groups, respectively, and then optically scanning the images to obtain fluorescence intensity and analyzing the results. . Gene expression is determined according to the ratio of the two fluorescence intensities (Somasundaram, K. et al. , Genomics Proteomics I: 1-10, 2002). In combination with recent research on toxic genomics (Toxicogenomics), an advanced technique using DNA macroarray technology, high-throughput expression in specific tissues or cell lines by all chemicals, including pharmaceuticals and new drug candidates, as well as representative environmental pollutants Analysis and quantitative analysis of expression patterns of genes are now possible. Accordingly, by analyzing the frequency of expression of specific genes in specific cells, it is possible to discover genes related to the adverse effects of drugs and the harmful effects of environmental pollutants. Molecular mechanisms will be understood, and further, the detection and identification of substances that cause toxicity and side effects will be possible.

Accordingly, the present inventors observed and analyzed a gene expression profile of Mirex in human liver cancer cells HepG2 cell line using oligomicroarray accumulated with about 45,000 human genes, and performed other residual organic pollutants 3 performed by the present inventors. Comparative analysis of gene expression profiles by species to find a gene that is specifically overexpressed only by Mirex to establish a biomarker capable of specifically detecting Mirex and a method for identifying the exposure using the present invention Completed.

It is an object of the present invention to provide a method for confirming whether or not to expose mirex using a biomarker specifically overexpressed by exposure of mirex, and to provide a kit for confirming whether or not to expose mirex using the biomarker.

In order to achieve the above object, the present invention provides a biomarker for confirming the exposure to mirex, characterized in that the expression changes specifically by exposure to mirex.

In another aspect, the present invention provides a DNA microarray chip for identifying whether or not the Mirex-specific exposure of the oligonucleotide or its complementary strand molecules containing all or part of the biomarker gene sequence.

In addition, the present invention provides a method for confirming whether or not the Mirex-specific exposure using the biomarker.

In addition, the present invention provides a search kit for identifying mirex-specific exposure.

The biomarker for confirming whether or not a mirex-specific exposure of the present invention and a verification method using the same are useful for determining the monitoring and risk of mirex using a reaction gene selected through a DNA microarray chip as a biomarker. It can be usefully used as a tool for identifying specific mechanisms of toxic effects caused by.

1 is a graph illustrating the cytotoxicity of mirex human liver cancer cell line.
Figure 2 is a diagram showing the results of analyzing the gene expression of the human liver cancer cell line treated with Mirex using a microarray chip.
Figure 3 is a diagram showing the results of the overall analysis of the gene expression after the treatment of a total of four persistent organic pollutants using a microarray chip.
Figure 4 is a graph comparing the microarray results of SULT1E1, a gene whose expression is increased by 1.5 times or more by Mirex alone, without increasing expression in the other three residual organic pollutants (Toxaphene DATA is not expressed on a chip). Did).

Hereinafter, the present invention will be described in detail.

The present invention provides a biomarker for confirming whether or not a Mirex-specific exposure is characterized by causing a change in expression specifically by exposure of a Mirex.

The biomarker is a gene whose expression is increased more than 1.5 times compared to the control group not exposed by Mirex exposure, and gene expression profile by three other residual organic pollutants in Table 1 of 45,000 genes. In comparison analysis, it consists of one gene whose expression changes only by Mirex.

List of 4 persistent organic pollutants Material name Persistent Organic Pollutants
(POPs) Chlordane Hexachlorobenzene Mirex Toxaphene

The present invention provides a biomarker which is selected from the group consisting of:

Genebank NM_005420 (SULT1E1, sulfotransferase family 1E, estrogen-preferring, member 1).

The present inventors, in order to find a biomarker for confirming whether or not Mirex-specific exposure, Mirex was treated to the HepG2 cell line of human liver cancer cells to confirm cytotoxicity. As a result, the Mirex was confirmed to be toxic to human liver cancer cell lines (see FIG. 1), and the concentration of Mirex was determined based on the experiment. Thereafter, Mirex was treated to human liver cancer cell line at the determined concentration, mRNA was isolated from the cell line treated with the substance, and cDNA was synthesized and labeled with fluorescent material (Cy5), and the drug-treated control group was labeled with Cy3. It was. The fluorescently labeled cDNA was hybridized with a 4 × 44 k oligomicroarray chip, Human whole genome microarray (Agilent, USA), and analyzed for differences in gene expression patterns by scanning fluorescent images (see FIG. 2). In the analysis, when the Cy5 / Cy3 ratio was 1.5 times or more, the expression was classified as increased gene, and when it was 0.66 or less, it was classified as the expression reduced gene. As a result, it was confirmed that the expression of the increased gene was 2.08% (945 out of 45,220 genes) and 0.79% (356 of the 45,220 genes) decreased in expression. At this time, one gene of which expression was specifically increased was selected only in Mirex when compared with the gene expression profile of three other residual organic pollutants. Although the gene has been reported to be involved in causing hepatotoxicity and related diseases by other chemicals in other existing studies (Table 2), it is associated with toxicity in human liver cancer cells when the mirex used in the present invention is treated. There is no report.

Chemicals and Hepatotoxic Diseases Affecting SULT1E1 Expression Hepatotoxicity-Related Diseases Trigger Liver Cirrhosis Ethanol Liver Cirrhosis, Experimental Dimethylnitrosamine, Estradiol, Ethanol,
Genistein, Quercetin, Thioacetamide Liver Diseases Acetaminophen, Diethylnitrosamine,
Thioacetamide Liver Neoplasms Clofibric Acid, Diethylnitrosamine,
Diethylstilbestrol, Dimethylnitrosamine,
2-Acetylaminofluorene, Quercetin Liver Neoplasms, Experimental Diethylnitrosamine, Dimethylnitrosamine,
2-Acetylaminofluorene, Hexachlorobenzene,
Polychlorinated Biphenyls, Quercetin Carcinoma, Hepatocellular Diethylnitrosamine, Diethylstilbestrol,
Estradiol, Ethanol, 2-Acetylaminofluorene,
Genistein, Quercetin, resveratrol,
Thioacetamide Fatty Liver Ethanol, Ethinyl Estradiol

The present invention also provides a DNA microarray chip for confirming whether or not a mirex-specific exposure in which an oligonucleotide or a complementary strand molecule thereof including all or part of the biomarker gene sequence is integrated.

The oligonucleotide or its complementary strand molecule includes 18 to 30 nucleic acids of the biomarker gene, and preferably 20 to 25 nucleic acids, but is not limited thereto.

The mirex retrieval DNA microarray chip of the present invention can be produced by a method known to those skilled in the art. The method of manufacturing the microarray chip is as follows. In order to immobilize the searched biomarker as a probe DNA molecule on a substrate of a DNA chip, a micropipetting method or a pin-shaped spotter using a piezoelectric method is used. It is preferable to use the method, but the present invention is not limited thereto. In a preferred embodiment of the present invention, a microarray, which is a pin-shaped spotter, is used. The substrate of the DNA microarray chip is preferably coated with one active group selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde. It is not limited. In addition, the substrate may be selected from the group consisting of slide glass, plastic, metal, silicon, nylon membrane, and nitrocellulose membrane, but is not limited thereto, and in the preferred embodiment of the present invention, amino-silane may be used. Coated slide glass was used.

In addition, the present invention provides a method for confirming whether or not Mirex specific exposure using the biomarker of the present invention.

The present invention provides a method for identifying a Mirex specific exposure comprising the following steps:

1) treating the test compound to human somatic cells;

2) separating RNA from the experimental group cells treated with the test compound of step 1) and control cells not treated with the test compound;

3) synthesizing RNA of the experimental group and the control group of step 2) with cDNA and labeling the fluorescent substance of the experimental group and the control group, respectively;

4) hybridizing cDNA labeled with different fluorescent materials of step 3) with DNA microarray chips;

5) analyzing the reacted DNA microarray chip of step 4); And

6) confirming the expression level of the biomarker of the present invention in the analyzed data of step 5) compared to the control.

In the method of confirming the exposure, the somatic cell of step 1) is preferably used as a hepG2 cell line of human liver cancer cells, but is not limited thereto, and any cells derived from cells and tissues of human liver cells or human liver cancers It is possible.

In the method for confirming the exposure, the fluorescent material of step 3) is Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC), RITC (rhodamine-B-isothiocyanate) and rhodamine (rhodamine) It is preferable to be selected but not limited thereto, and any fluorescent material known to those skilled in the art may be used.

In the method for confirming the exposure, the DNA microarray chip of step 5) is preferably used, such as Whole Human Genome Oligo Microarray (Agilent, USA), but is not limited thereto, the overexpression of the human genome in the present invention Any microarray chip loaded with a gene (Table 4) can be used, and it is most preferable to use a DNA microarray chip produced by the present inventor. In addition, the analysis method of step 5) preferably uses GenePix 4.1 software (Axon Instruments, USA), but is not limited thereto, and analysis software known to those skilled in the art may be used.

In addition, the present invention provides a method for confirming exposure to Mirex comprising the following steps:

1) treating the test compound to human somatic cells;

2) separating RNA from the test cell treated with the test compound of step 1) and the control cell not treated with the test compound;

3) performing a real-time reverse transcript polymerase chain reaction (RT-PCR) on the RNA of step 2) using a primer pair capable of amplifying the biomarker gene of the present invention; And

4) confirming the expression level by comparing the gene product of step 3) with the control.

In the method for confirming the exposure, the somatic cell of step 1) is preferably used as a hepG2 cell line of human liver cancer cells, but is not limited thereto, and any cells derived from human liver or liver cancer cells and tissues may be used. It is possible.

In the above method, the primer of step 3) is complementary to the biomarker gene found in the present invention, and any primer that can amplify the biomarker can be used.

In addition, the present invention provides a kit for confirming whether or not a mirex specific exposure using the biomarker of the present invention.

Specifically, the kit of the present invention preferably includes a probe set of a biomarker gene or a primer set for amplifying the gene, but is not limited thereto.

Preferably, the probe set is performed by a microarray, but more preferably, a kit including the DNA microarray chip of the present invention is manufactured and performed.

The primer set is preferably performed by PCR, but is not limited thereto.

The kit of the present invention may additionally comprise human hepatocytes. The human hepatocyte is preferably HepG2, but is not limited thereto. Any human hepatocyte may be used as long as it is derived from cells and tissues of human liver cells or human liver cancer.

The kit of the present invention may additionally include a fluorescent material, and the fluorescent material is composed of a strepavidin-like phosphatease conjugate, a chemical fluorescence (chemiflurorensce) and a chemiluminescent material (chemiluminescent). Preferably, but not limited to selected from the group, in the preferred embodiment of the present invention used Cy3 and Cy5.

The kit of the present invention may additionally include a reaction reagent, which is a buffer solution used for hybridization, reverse transcriptase for synthesizing cDNA from RNA, cNTPs and rNTP (premixed or separated feed), fluorescent staining agent It may be composed of a labeling reagent, such as a chemical inducing agent, a washing buffer, and the like, but is not limited thereto. Any reaction reagents required for hybridization of DNA microarray chips known to those skilled in the art may be included.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Cell Culture and Chemical Treatment

<1-1> Cell Culture

HepG2 cells (Korea Cell Line Bank), which is a human liver cancer cell line, were cultured in DMEM medium (Gibro-BRL, USA) to which 10% FBS was added until 80% growth in 100 mm dish. The present inventors selected Mirex, a substance having ecosystem persistence, as one of the persistent organic pollutants through existing research and reports, and dissolved in ethanol. Vehicle concentration was less than 0.1% in all experiments.

<1-2> MTT assay and chemical treatment

MTT experiment using HepG2 cell line was performed by the method of Mossman et al. ( J. Immunol. Methods , 65, 55-63, 1983). Cells were treated with mirx dissolved in DMSO in DMEM medium (Gibro-BRL, USA) at 4 × 10 ⁵ / well cell counts in 24-well plates, and after 48 hours, MTT (3-4,5-dimethylthiazol-2, 5 mg / mL of 5-diphenyltetra zolium bromide) was mixed and added to the tube and incubated at 37 ° C. for 3 hours. Thereafter, the medium was removed and the formed formazan crystal was dissolved in 500 µl of DMSO. Transfer to a 96-well plate was aliquoted and the OD value was measured at absorbance 540 nm.

As a result of examining the cytotoxicity of Mirex in the HepG2 cell line, the concentration showing 20% survival rate (IC ₂₀ ) was 41.23 uM (FIG. 1), and the microarray experiments were performed by determining the concentrations.

Example 2 Microarray Experiment

<2-1> Isolation of Target RNA and Fluorescent Labeling

After dispensing HepG2 cells in a 100 mm dish at a concentration of 6 × 10 ⁶ cells / ml, the concentration of mirex determined in Example 1-2 was treated for 48 hours. Thereafter, the whole cells were separated from the treated cells using a trizol reagent (Invitrogen life technologies, USA) according to the manufacturer's method, and purified using the RNeasy mini kit (Qiagen, USA). Genomic DNA was removed using RNase-free DNase set (Qiagen, USA) during RNA purification. The amount of each total RNA sample was measured by spectrophotometer and the concentration was confirmed by Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and agarose gel electrophoresis.

<2-2> Labeled cDNA Preparation

CDNA was prepared using the total RNA of the Mirex treated group obtained in Example 2-1 for oligo microarray analysis. The total RNA obtained above was mixed with 30 μg of oligo (dT) primer and 2 μg (1 μg / μl), reacted at 65 ° C. for 10 minutes, and then annealed in ice. Reagents were mixed as shown in Table 3 below for reverse transcription of the annealed RNA.

Configuration Volume (μl) 5X first strand buffer 6 dNTPs 0.6 0.1 M DDT 3 SuperScript II enzyme 3 Cy-3 or Cy-5 dUTP 2

Total RNA isolated from control HepG2 cell line was labeled with Cy3-dUTP (green), RNA isolated from Mirex-treated HepG2 cell line was labeled with Cy5-dUTP (red). At this time, the two samples were mixed and purified using a Microcon YM-30 column (Millipore, USA).

<2-3> hybridization reaction

Hybridization and washing procedures were carried out according to the instructions of Genome Co., Ltd .. Hybridization was performed in a 62 ° C. oven for 12 hours. 44 k Whole Human Genome Oligo Microarray (Agilent, USA) was used as the DNA microarray chip. After washing (washing in 2 × SSC / 0.1% SDS for 2 minutes, washing in 1 × SSC for 3 minutes, 0.2 × SSC for 2 minutes), the slides were dried by centrifugation at 800 rpm for 3 minutes.

<2-4> Fluorescence Image Acquisition

Hybridization images on the slides were scanned with the Genepix 4000B (Axon Instruments, USA). Chips washed with unbound genes were obtained with a fluorescence image using a laser fluorescence scanner. In this case, the green fluorescence image in the control group, the red fluorescence image represents the activity level of the gene specifically expressed only in the experimental group, and the yellow fluorescence image is the complementary color of green and red, which means that there is no significant difference between the two groups. Scanned images were analyzed with GenePix 4.1 software (Axon Instruments, USA) to obtain gene expression rates. Marker genes for mirex were selected from the data thus obtained (FIG. 2).

As a result, among the approximately 45,000 genes present on the oligo chip, 2.08% (945 out of 45,220 genes) were found to have increased expression of Cy5 / Cy3 by 1.5 times or more by Mirex. The reduced gene was found to be 0.79% (356 out of 45,220 genes).

At this time, when comparing the gene expression profile by the other three types of persistent organic contaminants (Fig. 3), only one gene whose expression was specifically increased was selected (Fig. 4). There is no report that the gene is associated with toxicity in human liver cancer cells when treated with the mirex used in the present invention.

Gene whose expression changes specifically by Mirex Registration Number Gene abbreviation Gene name Ratio of medians NM_005420 SULT1E1 sulfotransferase family 1E,
estrogen-preferring, member 1 1.57

As described above, the present invention can be usefully used to develop a microarray chip or kit to determine the risk and the monitoring of Mirex.

Claims (9)

Genebank No. NM_005420 (SULT1E1, sulfotransferase family 1E, estrogen-preferring, member 1) Oligonucleotides or complementary strand molecules that are all or a fragment of the gene consisting of 18 to 30 nucleic acid sequences of the gene Integrated DNA microarray chip for exposure to Mirex.
1) separating RNA from test subject-derived human somatic cells and control somatic cells;
2) labeling the experimental group and the control group with different fluorescent substances while synthesizing RNA of the experimental group and the control group of step 1) with cDNA;
3) hybridizing cDNA labeled with different fluorescent materials of step 2) with the DNA microarray chip of claim 1;
4) analyzing the reacted DNA microarray chip; And
5) comparing the expression level of the gene integrated in the DNA microarray chip in the analyzed data with a control group to determine whether the exposure to Mirex.
The method of claim 2, wherein the human somatic cells of step 1) are human hepatocytes or cells of human liver cancer.
The method of claim 3, wherein the human liver cancer cells are HepG2.
The method of claim 2, wherein the fluorescent material of step 3) is selected from the group consisting of Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate (RITC), and rhodamine (rhodamine). How to check whether exposure to Mirex.
Genebank NM_005420 (SULT1E1, sulfotransferase family 1E, estrogen-preferring, member 1) Probe set of genes or exposure to mirex comprising primer sets that amplify the genes Identification kit.
The kit for confirming exposure to mirex according to claim 6, wherein the probe set is performed by a microarray.
The kit for confirming exposure to mirex according to claim 6, wherein the primer set is performed by PCR.
The kit according to claim 6, further comprising human hepatocytes or liver cancer cells.

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