KR101567841B1 - Biomarker for detecting volatile organic compounds and method for detecting volatile organic compounds using the same - Google Patents

Abstract

The present invention relates to a biomarker for detecting a new volatile organic compound and a method for detecting a volatile organic compound using the biomarker.

According to the method of the present invention, a biomarker gene for detecting a volatile organic compound, which can be used in a toxicity problem of trace amounts and long-term exposure, which can not be solved by conventional classical risk assessment methods, .

In addition, the biomarker gene selected according to the method of the present invention can be easily used for the purpose of detecting volatile organic compounds.

Volatile organic compounds, biomarkers

Description

TECHNICAL FIELD [0001] The present invention relates to a biomarker for detecting a volatile organic compound and a method for detecting a volatile organic compound using the biomarker.

The present invention relates to a biomarker for detecting a new volatile organic compound and a method for detecting a volatile organic compound using the biomarker.

Volatile Organic Compounds (VOCs) are volatile organic compounds (VOCs) that volatilize in the air and generate odor and ozone. In Korea, hydrocarbons such as petrochemicals and organic solvents with a vapor pressure of 10.3 kilopascals It is managed as a substance to be designated as a notification. In the United States and Europe, it is defined as an organic compound causing NOx and photochemical oxidation reaction by the sunlight in the atmosphere to increase the ozone concentration on the surface to cause smog phenomenon.

Volatile organic compounds are carcinogens that cause damage to the nervous system through skin contact or inhalation. These volatile organic compounds often cause bad odors even at low concentrations, and the compounds themselves are directly harmful to the environment and human body, or participate in photochemical reactions in the atmosphere and generate secondary pollutants such as photochemical oxides.

In other words, the most important role of volatile organic compound (VOC) in the atmosphere exists as NOx and reacts with OH radical (Radical) to generate ozone, which becomes a cause of photochemical oxidant.

_{VOC + 2NO + 2O 2 → H} 2 O + 2NO 2 + R'C (O) R "

NO ₂ + hv → NO + O

_{O + O 2 + M → O} 3 + M

∴ VOC + 3O ₂ ➝H ₂ O + O ₃ + R'C (O) R "

Recently, as environmental pollution becomes serious due to rapid industrialization, it is necessary to have an effective analysis system capable of early identification and analysis of the risk of trace amounts of environmentally harmful substances. Especially, exposure to environment- The evaluation of human health damage is becoming an important environmental technology. Recent hazard assessment techniques focus on identifying low-dose exposure effects that can reflect human exposure levels in a realistic living environment, rather than relying on past toxicity testing results at high doses, And it is evolving as a method of setting management standards in consideration of the level.

Classically, in order to evaluate environmentally harmful substances, cells are cultured in vitro, ie, on a cell culture plate such as a 96-well plate, treated with toxic substances, Biochemical, hematologic, and pathologic observations were performed by detecting the number of cells through absorbance or directly in vivo, that is, by conducting animal experiments. This method consumes many samples and time, and there are ethical problems that come from animal experiments.

An E-screen assay can be used to assess the toxicity of toxic substances. Soto et al. Developed a method for evaluating the estrogenicity of environmentally harmful substances to be examined by in vitro method. The effect of chemical proliferation on MCF-7 cells (bus cell) , Which is an assay method based on gene expression induced by estrogen as a method for evaluating the estrogenic activity of a chemical substance, and an experiment is carried out using cloned MCF-7 Bus cells.

The estrogen receptor binding assay is a method recommended by the US EPA Tier 1 Screening in vitro method of EDSTAC. It is a method necessary to reveal the molecular mechanism of the effect of estrogen activity. It can be easily and quickly screened .

Uterotrophic assay is a method of indirectly assessing estrogenic activity by measuring the increase in uterine tissue mass induced by estrogen. Uterotrophic assays are recommended by the US EPA in the EDSTAC Tier 1 Screening in vivo method and are often referred to as the "gold standard" method. The uterine hypertrophic response test has been widely used for a long period of time in assessing etrosogenicity. However, since the route of administration and detailed experimental methods are not standardized, there is a difference between the experimental groups, and international efforts are being made to standardize the experimental methods.

A number of studies have been conducted worldwide to predict the risks of these environmentally harmful substances, including the ability to screen large quantities of DNA chips using microarray technology to assess risks to toxic and unknown substances Technology is considered to be very useful, and in the advanced countries such as the United States, a lot of efforts are being made for the development of related technologies at the national level. As part of the development of related technologies, there is a lot of progress in the fields of DNA chip technology and protein mass screen technology as a technology for predicting risk. In the case of DNA chips, technology has evolved to such an extent that expression screens for almost all known genes in the species are available.

In order to predict the toxicity, many analytical approaches have been made to predict the toxicity, because there has not been developed a clear analysis method for this technology development and much effort to develop the toxic prediction technology using the technology such as DNA chip to date. In the case of DNA chip, despite the technology capable of screening the expression of all known genes, there have been reported problems with differences in analysis results according to the theory of comparative analysis of gene expression in analysis. Also, (Russel S. Thomas 2001). In addition, the results of the present study indicate that the accuracy of the analysis method using a small number of specific genes selected by selecting specific genes is rather high.

In advanced countries such as the US, Japan, and the EU, long-term strategies such as research on environmental distribution of toxic substances, exposure to human body, development of test methods using new toxic genetic technologies for toxicological effects evaluation, . However, since there is no internationally accepted detection and testing method to date, it is difficult to compare and evaluate test data among research institutes. Therefore, it is required to test the environmentally harmful substance test method which can be commonly accepted by national institutions and related materials producing organizations. The approach to environmentally harmful substances is not merely the concept of "there is no", but it needs to be precisely prevented from exposure to substances through analysis of precise mechanism of harmful substances based on high technology. And the introduction of toxic genomic technology is urgently required in the study.

Conventional methods have the disadvantage that toxicity which is remarkable can be evaluated by examining the change when the human body is exposed to environmentally harmful substances, but it is not always effective in predicting toxicity which is not yet shown or in the risk evaluation which is extremely weak. Therefore, the present invention provides a biomarker gene that can be used for predicting toxicity, that is, for detecting environmental harmful substances, especially volatile organic compounds, that exhibit potential toxicity through the result of toxic dielectric technology. Also, a method for detecting a volatile organic compound using such a new biomarker is provided.

In order to search for a gene having a significant expression change in a cell line upon administration of a representative known volatile organic compound using the entire microarray, the inventors of the present invention found that a gene showing a significant expression change in each of two or more specific volatile organic compounds It was found that it is useful for the detection of volatile organic compounds remarkably.

Therefore,

(a) independently treating two or more known volatile organic compounds (Volatile Organic Compounds) in a cell line to detect respective gene expression profiles;

(b) detecting a gene expression profile in the same cell line as in step (a) when the same conditions as in step (a) are maintained without treating the volatile organic compound in the same cell line; And

(c) comparing the profiles of steps (a) and (b) to select a gene showing a significant change in the same expression profile in both of the two or more volatile organic compounds, and judging the gene as a biomarker gene for detecting a volatile organic compound A method for screening a biomarker gene for detecting a volatile organic compound.

The present invention also provides biomarker genes for the detection of volatile organic compounds selected by the screening method of the present invention.

In addition,

(a) treating the cell line with a sample suspected of containing a volatile organic compound;

(b) detecting the expression profile of the biomarker gene of claim 4 before or after the treatment of step (a) in said cell; And

(c) comparing the profile detected in step (b) with the biomarker gene expression profile of the cell line that has undergone the same steps as in steps (a) and (b) except that the volatile organic compound is not treated, And judging that the sample showing the difference contains the volatile organic compound.

Hereinafter, the present invention will be described in more detail.

In one aspect of the invention,

(a) independently treating two or more known volatile organic compounds (Volatile Organic Compounds) in a cell line to detect respective gene expression profiles;

(b) detecting a gene expression profile in the same cell line as in step (a) when the same conditions as in step (a) are maintained without treating the volatile organic compound in the same cell line; And

(c) comparing the profiles of steps (a) and (b) to select a gene showing a significant change in the same expression profile in both of the two or more volatile organic compounds, and judging the gene as a biomarker gene for detecting a volatile organic compound A method for screening a biomarker gene for detecting a volatile organic compound.

The steps (a) and (b) may be carried out by any means capable of screening all possible gene expressions of the cell. Preferably, a whole genome oligonucleotide microarray capable of easily detecting an expression pattern can be used.

The cell line can be any cell capable of responding to environmental pollutants including volatile organic compounds. Preferably, the cell line is a human-derived cell line, more preferably a human blood-derived cell line such as HL-60, but is not limited thereto.

The two or more known volatile organic compounds usable in the present invention are not particularly limited. The more thoroughly the adverse effects of volatile organic compounds are verified, the more desirable. The volatile organic compound is preferably selected from substituted or unsubstituted benzene, toluene, xylene and formaldehyde series. Preferably, the two or more known volatile organic compounds of the present invention are selected from the group consisting of substituted or unsubstituted benzene, toluene, xylene, and formaldehyde.

In the examples of the present invention, the known volatile organic compounds of the present invention were assayed for IC ₅₀ values by MTT assay using ethylbenzene and trichlorethylene, and the resulting concentrations were treated on HL-60 cells, The changes in the profile were compared with the untreated control group.

In the method of the present invention, in order to screen a biomarker gene showing a change in the gene expression profile, the results obtained by treating two or more kinds of the volatile organic compounds are collected, and only genes showing similar changes in the expression profile are selected. Thus, a generally available biomarker gene detection system with high reliability compared to the results obtained by treating only one volatile organic compound is disclosed.

In another aspect of the present invention, there are provided biomarker genes for detecting new volatile organic compounds.

The biomarker gene for detecting a volatile organic compound of the present invention may be any gene which is screened by the method of the present invention. The present inventors have detected 16 biomarker genes as a result of treating a specific volatile organic compound by the above method (see FIGS. 7 and 8), but the genes that can be detected using this method are not limited thereto.

In one embodiment of the present invention, two kinds of volatile organic compounds, which are applied to the whole genomic oligonucleotide microarray chip used, are compared with the non-treated group. Genes showing the above expression change patterns in common for the compounds were screened and shown in FIGS. 7 and 8. FIG.

In another aspect of the present invention,

(a) treating the cell line with a sample suspected of containing a volatile organic compound;

(b) detecting the expression profile of the biomarker gene of claim 4 before or after the treatment of step (a) in said cell; And

(c) comparing the profile detected in step (b) with the biomarker gene expression profile of the cell line that has undergone the same steps as in steps (a) and (b) except that the volatile organic compound is not treated, And judging that the sample showing the difference contains the volatile organic compound.

Since the biomarker gene of the present invention is screened from the whole genomic oligonucleotide microarray chip in the sample suspected of containing a volatile organic compound in the present invention, the presence of the volatile organic compound can be effectively detected by detecting the change in the expression profile in the same cell Can be determined.

MRNAs in the treated and untreated samples were analyzed by using a method of hybridizing the whole cDNA labeled with fluorescent Cy3-dUTP and Cy5-dUTP to the microarray chip In the control group, red indicates the degree of activity of the gene specifically expressed in the experimental group, and yellow indicates the green and red complementary colors, meaning that there is no significant difference between the two groups. The overall intensities are normalized using correlation coefficients obtained from the percentage of housekeeping genes. Identification of the expression pattern of the biomarker gene from the expression profile data thus obtained can easily identify the presence of volatile organic compounds in the sample. Therefore, the genes of the present invention can be usefully used to search for and determine volatile organic compounds by the above method.

The above method is merely an illustrative example, and the method for determining the presence or absence of volatile organic compounds by measuring changes in the gene expression profile using the biomarker gene of the present invention by a classical method is also within the scope of the present invention.

According to the method of the present invention, a biomarker gene for detecting a volatile organic compound, which can be used in a toxicity problem of trace amounts and long-term exposure, which can not be solved by conventional classical risk assessment methods, .

In addition, the biomarker gene selected according to the method of the present invention can be easily used for the purpose of detecting volatile organic compounds.

In addition, the biomarker gene screening method and the biomarker gene of the present invention are useful for ecotoxicogenomics studies considering the environmental characteristics of a substantial biological reaction.

Hereinafter, the present invention will be described in more detail by way of examples. However, these embodiments are not intended to limit the scope of the invention.

< Example >

Example  1: Cell culture and chemical treatment

1-1 Cell Culture

HL-60 cells (obtained from Korea Cell Line Bank), a human myelogenous leukemia cell line, were cultured in T-75 cell culture flask with confluency of about 80% using RPMI medium (Gibro-BRL, USA) supplemented with 10% FBS Lt; / RTI &gt; until grown. In order to screen biomarker genes, ethylbenzene and trichlorethylene, which are volatile organic compounds (VOCs) and substances known to have carcinogenic properties, were selected as the known volatile organic compounds based on previous studies and reports. They were dissolved in DMSO . The vehicle concentration was less than 0.1% in all experiments.

1-2 Cytotoxicity Test MTT assay ) And chemical treatment

MTT experiments using the HL-60 cell line were performed using the method of Mossman et al . ( J. Immunol . Methods , 65: 55-63, 1983). Cells were treated with ethylbenzene and trichlorethylene dissolved in DMSO in RPMI medium (Gibro-BRL, USA) at a cell density of 1 × 10 ⁶ cells / ml in 15 ml tubes and MTT (3-4, 5- dimethylthiazol-2, 5-diphenyltetra zolium bromide) was added to the tube and incubated at 37 ° C for 3 hours. The medium was then removed and the formazan crystal formed was dissolved in 500 μl of DMSO. Transferred to 96-well plates and aliquoted and the OD value measured at an absorbance of 540 mm.

As a result, the 50% survival rate (IC50) was 53.86 mM, 0.9854 mM and 4.112 mM, respectively, in the HL-60 cell line (FIGS. 1A and 1B) , Microarray experiments were performed using the above concentrations.

Example  2: All RNA  detach

To 1 × 10 ⁶ cell / ㎖ after dispensing the concentration HL-60 cells in T-75 flasks, the ethylene in Example 1-2 concentrations of ethyl benzene and trichloro determined in treated for 3 hours. Then, total RNA was isolated from the treated cells according to the manufacturer's instructions using a trizol reagent (Invitrogen life technologies, USA) and purified using an RNeasy mini kit (Qiagen, USA). Genomic DNA was removed using an RNase-free DNase set (Qiagen, USA) during RNA purification. The amount of each total RNA sample was determined by nano-drop and the quality was confirmed by Agilent bioanalyzer 2100 (Agilent technologies).

Example  3: Microarray

3-1 Labeled  all cDNA  Produce

For the microarray analysis, cDNA was prepared using total RNAs from reference control and chemical treatment groups. Labeling and hybridization were performed using a slight modification of the MICROMAX direct cDNA microarray system (PerkinElmer life sciences, MA, USA). 30 to 50 μg of the total RNA prepared above was mixed with 2 μg (1 μg / μl) of the oligo (dT) primer, reacted at 70 ° C for 5 minutes, and immediately annealed by ice. Next, reagents were mixed as shown in the following table to perform a reverse transcription (RT) reaction of the annealed RNA.

RNA isolated from HL-60 cells treated with volatile organic compounds was labeled with Cy3-dUTP (NEN, MA, USA), and RNA isolated from untreated cells was labeled with Cy5-dUTP (NEN). At this time, the two colors were mixed and purified using alcohol precipitation.

3-2 Hybridization  reaction

Hybridization was carried out in a 62 [deg.] C hybridization oven for 12 hours. After washing (2 x SSC / 0.1% SDS at 62 캜 for 5 minutes, 0.1 x SSC / 0.1% SDS at room temperature for 10 minutes, 0.1 x SSC at room temperature for 1 minute), the slide was centrifuged at 3000 rpm for 20 seconds and dried.

3-3 Fluorescence image acquisition

Hybridization images on the slides were scanned with GenePix 4000B (Axon instruments). After washing to remove unbound genes, the chip was read by a laser fluoroscopy scanner. In this case, red indicates the degree of activity of the gene specifically expressed in the experimental group only in the control group, and yellow indicates a complementary color between green and red. Scanned images were analyzed with GenePix 5.1 software (Axon Instruments, CA, USA) to obtain gene expression ratios. Overall intensities were normalized using correlation coefficients obtained from the proportion of positive control genes. From the data thus obtained, the expression profiles of the ethylbenzene and trichlorethylene used as the known volatile organic compounds in the above Examples were identical to each other, that is, the genes exhibiting the same significant increase or the same significant decrease Respectively. (See Figs. 2A to 6)

The two types of volatile organic compounds treated with the whole genomic oligonucleotide chip used in the above-mentioned examples were compared with the non-treated group. And the gene showing the above expression change pattern was selected and shown in FIG. 7 and FIG.

The genes that are significantly increased in expression of both volatile organic compounds are ISG15 ubiquitin-like modifier, tumor necrosis factor (ligand) superfamily, member 10, prion protein, malic enzyme 1 (NADP (+ In the present study, we investigated the expression of PPARγ, PPARγ, PPARγ, PPARγ, PPARγ, PPARγ, PPARγ, PPARγ, binding protein, alcitonin / calcitonin-related polypeptide (alpha), C-reactive protein (pentraxin-related), BCL2-associated X protein, chromosome 7 open reading frame 40 and fibroblast growth factor receptor 1.

The results indicate that volatile organic compounds including ethylbenzene and trichlorethylene are useful for screening biomarker genes for use in detecting volatile organic compounds at gene expression levels and that the genes are useful as biomarker genes for detection of volatile organic compounds .

1 is a graph showing the cytotoxicity of ethylbenzene and trichlorethylene in the HL-60 cell line measured by MTT assay. In the examples, microarray experiments were carried out at concentrations of 0.9854 mM and 4.112 mM, respectively, showing 50% survival rate (IC50).

Figure 2a is a photograph of the inside of the microarray before the experiment (Quality Check (Syto61 test)).

2B shows a microarray hybridization image obtained by treating a cDNA chip of total RNA with a volatile organic compound.

FIG. 3 shows the result of plotting for the correlation plot after confirming the fluorescence signal intensity after the treatment with the volatile organic compounds labeled with Cy3 and Cy5 and hybridization with the untreated HL-60 cell gene.

Figure 4 shows the plotting results for a normalization box-plot.

FIG. 5 is a result of a microarray reliability check. FIG.

6 is a photograph showing the results of hierarchical clustering after performing microarray experiments using ethylbenzene and trichlorethylene.

FIG. 7 is a graph showing genes whose expression patterns are commonly changed by ethylbenzene and trichlorethylene as volatile organic compounds. The up-regulated genes on the basis of 1.5 times in each substance Respectively.

FIG. 8 shows the genes whose expression patterns are commonly changed by ethylbenzene and trichlorethylene, which are volatile organic compounds, and genes down-regulated on the basis of 1.5 times of each substance.

<110> IUCF-HYU <120> BIOMARKER FOR DETECTING VOLATILE ORGANIC COMPOUNDS AND METHOD FOR          DETECTING VOLATILE ORGANIC COMPOUNDS USING THE SAME <160> 16 <170> Kopatentin 1.71 <210> 1 <211> 70 <212> DNA <213> Homo sapiens <400> 1 ggcgggcgga gctaagggcc tccaccagca tccgagcagg atcaagggcc ggaaataaag 60 gctgttgtaa 70 <210> 2 <211> 69 <212> DNA <213> Homo sapiens <400> 2 ctctcaagta gtgtatcaca gtagtagcct ccaggtttcc ttaagggaca acatccttaa 60 gtcaaaaga 69 <210> 3 <211> 69 <212> DNA <213> Homo sapiens <400> 3 gtccttaggc ttacaatgtg cactgaatcg tttcatgtaa gaatccaaag tggacaccat 60 taacaggtc 69 <210> 4 <211> 70 <212> DNA <213> Homo sapiens <400> 4 cacagtttat cctgaaccgc aaaacaaaga agcatttgtc cgctcccaga tgtatagtac 60 tgattatgac 70 <210> 5 <211> 69 <212> DNA <213> Homo sapiens <400> 5 tattatataa aggtggtggg caaaaacaat gtaaggagcc tttccagtta tcttgagttg 60 cagctctgt 69 <210> 6 <211> 69 <212> DNA <213> Homo sapiens <400> 6 tagctcaggc ttattcacat gatggcttca ggattccaaa gagagtgaga gtagaagctg 60 aaagacttc 69 <210> 7 <211> 69 <212> DNA <213> Homo sapiens <400> 7 tctccaagtc caccagtctc tgaaattaga acagtaggcg gtatgagata atcaggccta 60 atcatgttg 69 <210> 8 <211> 69 <212> DNA <213> Homo sapiens <400> 8 aagccatctt cctgcctact ggatgcttac agtgactgtg gatacggggg ttccctttcc 60 ccattcagt 69 <210> 9 <211> 69 <212> DNA <213> Homo sapiens <400> 9 cacaaagggt tgggagctga tgaaactcac aaatgatggt aggaagaagc tctcgacaat 60 acccgttgg 69 <210> 10 <211> 70 <212> DNA <213> Homo sapiens <400> 10 gtccaagatg ctaatggcct ctttatggca gatcgtgcag gtcgcaagtg gatcctgact 60 gacaaagcca 70 <210> 11 <211> 70 <212> DNA <213> Homo sapiens <400> 11 taatggatgt gcaggactca acacatgtga gctgtaaact ctacaaaggg ctgtcggatg 60 cactgatctg 70 <210> 12 <211> 70 <212> DNA <213> Homo sapiens <400> 12 caaaagttct cccccttcct ggctctcagc atcttggtcc tgttgcaggc aggcagcctc 60 catgcagcac 70 <210> 13 <211> 70 <212> DNA <213> Homo sapiens <400> 13 tcagcgcctg agaatggagg taaagtgtct ggtctgggag ctcgttaact atgctgggaa 60 acggtccaaa 70 <210> 14 <211> 70 <212> DNA <213> Homo sapiens <400> 14 ctgagcagat catgaagaca ggggcccttt tgcttcaggg tttcatccag gatcgagcag 60 ggcgaatggg 70 <210> 15 <211> 70 <212> DNA <213> Homo sapiens <400> 15 ttgaggtatc atcatgaggt cgtttttaag ttcagtacag atgaggggcc caaggtcaga 60 attatgtgtc 70 <210> 16 <211> 70 <212> DNA <213> Homo sapiens <400> 16 cggtttggtc tgttttgcct tcacccataa gcccctcgca ctctggtggc aggtgccttg 60 tcctcagggc 70

Claims (8)

delete delete delete delete delete (a) treating the cell line with a sample suspected of containing a volatile organic compound; (b) detecting the expression profile of the biomarker gene in the cell before and after the treatment of step (a); And (c) comparing the profile detected in step (b) with the biomarker gene expression profile of the cell line that has undergone the same steps as in steps (a) and (b) except that the volatile organic compound is not treated, Determining that the sample showing the difference includes the volatile organic compound, Wherein the volatile organic compound is ethylbenzene and trichlorethylene, The biomarker gene includes a homo sapiens ISG15 ubiquitin-like modifier, a homo sapiens tumor necrosis factor (ligand) superfamily, a member 10, a gene homologue NM_183079 (homo sapiens prion protein) (Homo sapiens sulforedoxin 1), Gene registration number NM_003330 (Homo sapiens malic enzyme 1, NADP (+) - dependent, cytosolic), Gene registration number NM_005437 sapiens thioredoxin reductase 1), Homo sapiens X-box binding protein 1, Homo sapiens membrane metallo-endopeptidase, Homo sapiens nuclear respiratory factor 1, NM_004774 (PPAR binding protein), homo sapiens calcitonin-related polypeptide alpha (NM_001741), gene registration number NM_000567 (Homo sapiens C-reactive protein, pentracin-related, homo sapiens BCL2-associated X protein, chromosome 7 open reading frame 40 and homo sapiens fibroblast growth factor receptor 1 (NM_023110) &Lt; / RTI &gt; wherein the volatile organic compound is selected from the group consisting of: delete delete

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