KR101488671B1 - Biomarkers for diagnosing the addiction of trichloroethylene - Google Patents

Abstract

The present invention relates to a biomarker protein for diagnosing trichlorethylene poisoning. More specifically, the present invention relates to a protein biomarker whose expression is markedly increased or decreased in a test group of trichlorethylene exposure compared with normal cells, and a diagnostic kit using the same .

Trichlorethylene, biomarkers, toxicity, poisoning

Description

TECHNICAL FIELD [0001] The present invention relates to a biomarker for diagnosing toxicity of trichlorethylene,

The present invention relates to a biomarker capable of diagnosing trichlorethylene poisoning, which is a kind of toxic substance, and more specifically, a protein biomarker that significantly increases or decreases expression in trichlorethylene exposure test group as compared with normal cells, And a diagnostic kit using the same.

Trichlorethylene is a kind of volatile organic compounds, also called trichloroethane, a colorless heavy mobile liquid having the formula C ₂ HCl ₃ and a molecular weight of 131.4. It has a specific gravity of 1.4556 (20 ° C), a melting point of -86.4 ° C, a boiling point of 86.9 ° C, a flash point of 32 ° C and an odor similar to chloroform. The toxic permissible concentration is defined as 50ppm and it is anesthetic, nonflammable, water-insoluble and miscible with alcohol, ether and other common organic solvents. Particularly, they are freely miscible with ethanol and less corrosive to metals. It has a vapor density of 4.5 and a vapor pressure of 100 mmHg (32 ° C). It is dissolved in ether, ethanol and chloroform. It decomposes slowly by moisture and light to generate hydrogen chloride.

Trichlorethylene can be used for de-oiling and cleaning of metal machinery parts, solvents (raw materials, dyes, paints, preservatives, sulfur, asphalt, pitch and cadmium), insecticides, degreasing cleaning of wool, leather, textile industry, , Refrigeration refrigerant, and the like. It is used to make seasonings by extracting oil from plant materials such as beans, coconut, and palm trees, caffeine-free food industry, and extracts of hops and spices. It is also used in the final water drying process during 100% ethanol production. Trichlorethylene was proven to be useful as a degreaser, but long term exposure in metallic bodies and reactivity at high temperatures are unstable and can condense or change when subjected to heat.

The exposure range of trichlorethylene is wide, and waste treatment areas throughout the United States include trichlorethylene as a common source of soil and groundwater. It is exposed when inhaled vapors of contaminated drinking water or contaminated soil or contaminated ground water near residential areas and when used as an anesthetic during prolonged surgery.

Trichlorethylene, together with other anesthetic gases, weakens the function of the central nervous system. In the 1930s and 1960s, trichlorethylene (Trimar ™ and Trilene ™) was used as an anesthetic in volatile gas in Europe and North America, and was used for purposes such as chloroform and ether. Trichlorethylene was superior in induction time and cost It has also been used as an alternative. However, as the neurotoxicity and neurological side effects caused by acute exposure to trichlorethylene appeared, use as an anesthetic was discontinued. Exposure to trichlorethylene results in symptoms similar to those of alcoholism, headache, dizziness, and loss of consciousness. Exposure to the liver and kidneys beyond the effects of the central nervous system at prolonged exposure is toxic.

Trichlorethylene was evaluated for carcinogenicity in the first animal experiment conducted in the 1970s. Exposure to trichlorethylene resulted in cancer in the liver and kidney of mice (Rhomberg, LR (2000) Dose-response analyzes of the carcinogenic effects of trichlorethylene in experimental animals. Environ. Health. Perspect., 108 (2), 343-358 .). There is an increasing number of studies on the infectivity in the workplace with trichlorethylene exposure, the relationship between trichlorethylene and cancer, and related evidence. Recent studies on the carcinogenic mechanism of trichlorethylene have reported that cancer is caused by metabolism occurring in the liver and studies on trichlorethylene exposure in experimental animals and humans have shown that there is an association between congenital heart defects (Scialli, AR and Gibb, H. (2005) Trichlorethylene exposure and congenital heart defects. Birth Defects Res. A Clin. Mol. Teratol., 255, 256-257). The relationship between levels of exposure and cardiac defects is not clear, but it is questionable whether it is consistent and may affect infertility as a result of animal testing. The International Agency for Research on Cancer (IARC) has identified this substance as Group 2A, a human carcinogen (IARC). IARC Monogr Eval Carcinog Risks Hum (1995) 63,75-158).

It is an object of the present invention to provide a biomarker for diagnosing toxicity of trichlorethylene which has high specificity.

In order to achieve the above object, the inventors of the present invention conducted studies to overcome the problems of the prior art, and as a result, they found that the two-dimensional electrophoresis technique significantly increased expression in the trichlorethylene treatment test group as compared with normal control cells, The inventors of the present invention have invented a biomarker for diagnosis using a protein which is remarkably lowered.

The biomarker for diagnosing trichlorethylene poisoning according to the present invention can sensitively and specifically detect the toxicity of trichlorethylene by detecting the change in the expression amount of at least one of the eight proteins.

The present invention provides a protein biomarker or immunogenic fragment thereof whose expression is markedly increased or decreased in the test group of trichlorethylene compared with normal cells.

The protein biomarker

The immunogenic fragment refers to a portion of the protein capable of producing a reactive antibody when injected into an external host.

The present invention provides an antibody that specifically binds to the biomarker protein or an immunogenic fragment thereof. The antibody of the present invention can be prepared according to methods well known to those skilled in the art.

The present invention provides a diagnostic kit characterized by diagnosing trichlorethylene poisoning by an increase or decrease in the amount of the biomarker protein as compared with a normal control group. The kit includes a kit for ELISA (Enzyme linked immunosorbent assay).

The present invention provides a method for diagnosing trichlorethylene poisoning through biomarker detection comprising contacting an antibody specifically binding to the biomarker protein with a biological sample selected from urine, blood, serum and plasma.

Further, the marker detection method of the present invention

a) providing a biological sample to be analyzed;

b) contacting the sample with an antibody that specifically binds to the one or more biomarker proteins;

c) quantitatively detecting the antigen-antibody complex; And

d) comparing the detection result of step c) with a normal control group.

In the present invention, the term "marker" is a substance capable of diagnosing trichloethylene intoxication cells differentiated from normal cells, and may be a polypeptide marker for the biomarker protein. These markers may be trichlorethylene- The expression is increased or decreased.

In the present invention, "diagnosis" means identifying the presence or characteristic of a trichlorethylene poisoning state.

The increase or decrease in the amount of the biomarker protein expression in the biological sample can be detected by confirming the amount of the protein. The process of separating the protein from the biological sample can be performed using a known process. The amount of the protein can be measured by various methods known to those skilled in the art .

In the present invention, the "biological sample" is a tissue or cell capable of detecting a difference in the level of the biomarker protein, and is preferably, but not limited to, urine, blood, plasma, serum and the like.

In the present invention, "measurement of protein level" is a process for confirming the expression level of the biomarker protein expressed or decreased in trichlorethylene intoxicated cells in a biological sample. Preferably, the antibody specifically binds to the biomarker protein To confirm the amount of protein.

"Antibody" refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to the biomarker protein, and includes both a polyclonal antibody, a monoclonal antibody, and a recombinant antibody.

Analysis methods for measuring protein levels using antibodies include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, ouchterlony immunodiffusion, rocket immunoelectrophoresis , Immunohistochemistry, immunoprecipitation, complement fixation assay, FACS, protein chip, and the like.

Antibody-antibody complex formation in the normal control group and the antigen-antibody complex formation in the suspected tricholethylene intoxication patient can be compared with each other and the increase in the biomarker protein expression level can be determined through the above analysis method. It is possible to diagnose the actual addiction of the patient.

The term "antigen-antibody complex" means a combination of the biomarker protein and the antibody specific thereto, and the amount of the antigen-antibody complex formed can be quantitatively measured through the signal label of the detection label. The detection label may be selected from the group consisting of an enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle, a redox molecule and a radioactive isotope, and is not limited to the substance described above. Examples of enzymes that can be used when the enzyme is used as a detection label include? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase or alkaline phosphatase, acetyl Cholinesterase, glucose oxidase, hexokinase, and the like, and is not limited to the range described above. Fluorescent substances include fluorescein, phycocyanin, fluorescamine, and the like, and are not limited to the substances described above. Examples of the ligand include, but are not limited to, biotin derivatives and the like. The luminescent material includes, but is not limited to, luciferin. The fine particles include, but are not limited to, colloidal gold. Examples of redox molecules include, but are not limited to, quinone, 1,4-benzoquinone, and hydroquinone. Radiation isotopes include, but are not limited to, ³ H, ¹⁴ C, and the like.

Determination of protein expression levels preferably uses an ELISA. ELISA is a direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of an antibody and an antigen attached to a solid support, an antibody against an antigen attached to a solid support, and another antibody recognizing the antigen And an indirect sandwich ELISA using a labeled secondary antibody that recognizes this antibody. More preferably, the antibody is attached to a solid support, the sample is reacted, and the labeled antibody recognizing the antigen of the antigen-antibody complex is adhered to produce an enzyme, or an antibody that recognizes the antigen of the antigen-antibody complex Is detected by a sandwich ELISA method in which a labeled secondary antibody is attached and the enzyme is developed. The degree of complex formation between the biomarker protein and the antibody can be confirmed to confirm the toxicity of trichlorethylene.

Alternatively, one or more antibodies against a biomarker may be arranged at a predetermined position on a substrate to use a protein chip immobilized at a high density. A method for analyzing a sample using a protein chip is to separate the protein from the sample, hybridize the separated protein with the protein chip to form an antigen-antibody complex, and read the protein to confirm presence or expression of the protein .

Alternatively, Western blotting is performed using an antibody against the biomarker. The whole protein is separated from the sample, and the protein is separated according to size by electrophoresis, and then transferred to the nitrocellulose membrane to react with the antibody. The amount of the generated antigen-antibody complex can be confirmed using the labeled antibody to confirm the poisoning.

Such detection methods include comparing the biomarker protein expression level of the normal control with the expression level of the marker protein in the trichlorethylene intoxicated cell. Protein levels can be expressed as absolute or relative differences in biomarker protein.

The present invention relates to a diagnostic kit for trichlorethylene poisoning comprising an antibody specifically binding to the biomarker protein.

In the present invention, since the eight kinds of biomarker proteins have been identified as trichlorethylene addiction markers, it is easy to produce antibodies by using techniques well known in the art. As is well known in the art to which the present invention pertains, a polyclonal antibody is obtained from an animal by injecting the biomarker protein antigen into the animal to obtain a serum containing the antibody. The animal can be produced using any animal host such as goat, rabbit, pig, etc. Monoclonal antibodies may be obtained from the hybridoma method (see Kohler & Milstein (1976) European J. Immunology 6 : 511-519) or the phage antibody library (Clackson et al. Nature , 352 : 624-628, 1991; Marks et al . , J. Mol. Biol. , 222 : 58, 1-597, 1991).

The hybridoma method uses cells of an immunologically appropriate host animal such as a mouse injected with a biomarker protein antigen, and the other uses a cancer or myeloma cell line. These two types of cells are fused by a method well known in the art such as polyethylene glycol, and then the antibody producing cells are proliferated by a standard tissue culture method. After obtaining a uniform cell population by subcloning by a limited dilution technique, hybridomas capable of producing antibodies specific for the biomarker protein are mass-cultured in vitro or in vivo according to standard techniques.

The phage antibody library method involves obtaining an antibody gene for a biomarker protein and expressing it in the form of a fusion protein on the surface of a phage to produce an antibody library in vitro and to prepare a monoclonal antibody Is a method of separating and manufacturing.

The antibody produced by the above method can be separated by electrophoresis, dialysis, ion exchange chromatography, affinity chromatography, or the like.

Antibodies included in the kits of the invention include functional fragments of antibody molecules as well as complete forms with two full length light chains and two full length heavy chains. The functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') ₂ , F (ab) ₂ and Fv.

The diagnostic kit of the present invention comprising an antibody that specifically binds to a biomarker protein is preferably a diagnostic kit for ELISA and can include an antibody specific for a control protein and can also detect an antigen- A reagent, such as a labeled secondary antibody, a chromophore, an enzyme conjugated to an antibody, and other substances capable of binding to the substrate or antibody thereof.

Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited thereto.

Experimental Method

<Protein Extraction>

For the proteomic analysis, a normal control group treated with corn oil, 1/10 LD ₅₀ (565 mg / kg) and The liver tissues were separated from the rats treated with 1/2 LD ₅₀ (2825 mg / kg) of trichlorethylene for 72 hours, disrupted with a homogenizer, and resuspended in 7M urea, 2M thiourea, 4.5% CHAPS {3 [(3-Cholamidopropyl ) dimethylammonio] -propanesulfonic acid}, and 100 mM DTE (dithioerythritol) in a 40 mM Tris-HCl buffer (pH 8.8). The shredded sample was centrifuged at 12,000 × g for 50 minutes at 15 ° C., and the resulting supernatant was used. The extracted proteins were stored at -70 ° C.

<Isoelectric point electrophoresis>

Total protein isolated from normal and experimental rat liver tissues was 1 mg, and 450 μl of rehydration solution {7M urea, 2M thiourea, 4.5% CHAPS, 0.25% ampholytes, 100 mM DTE} was used to dissolve the protein. Protein of each condition dissolved in 450 μl of rehydrated solution was injected into an IPG holder and immobilized dry strip (pH 3-10 NL, GE Healthcare Bio-Science) for 18 hours. 200V / 200 Vhrs, 500V / 500Vhrs, 1,000V / 1,000Vhrs, 8,000V / 13,500Vhrs and 8,000V / 100,000 Vhrs depending on the pH of the protein.

<Equilibrium>

The isoelectric point electrophoresis strips were subjected to primary equilibration for 20 minutes in primary equilibration buffer (6M urea, 0.375 M Tris-HCl (pH 8.8), 20% glycerol, 2% SDS, 25% acrylamide, 2 mM TBP) Secondary equilibration was performed for 20 minutes in a secondary equilibration buffer (6 M urea, 0.375 M Tris-HCl (pH 8.8), 20% glycerol, 2% SDS, 25% acrylamide, 2 mM TBP) for 20 minutes.

&Lt; SDS-polyacrylamide gel electrophoresis >

After making the 9% - 16% acrylamide gel, the strip with the isoelectric transfer was placed on the gel, fixed with 0.5% agarose gel, and then subjected to two dimensional electrophoresis. The protein was transferred according to the molecular weight by applying a current of 5 mA per gel for 2 hours and then applying 18 mA per gel for 10 hours.

&Lt; Gel dyeing and bleaching &

The developed gel was incubated with a Coomassie blue G-250 staining solution (34% methanol, 0.1% Coomassie G-250, 17% ammonium sulfate, 3% phosphoric acid) for more than 12 hours After staining, it was decolorized with 1% acetic acid.

<Gel image analysis>

For gel image analysis, the gels stained with UMAX powerLook 1100 were scanned and analyzed with ImageMaster 2D Platinum 6.0 (2D software, GE Healthcare Bio-Science). Three gels were analyzed per condition for accurate analysis.

<Nano LC-MS / MS analysis>

After in-gel digestion of each spot with trypsin for nano LC-MS / MS analysis, the peptides of the spots were analyzed with Agilent 110 Series Nano-LC and LTQ-Mass Spectrometry (Thermo Electron, San Jose, CA). Capillary columns (150 mm x 0.075 mm) used in the LC-MS / MS analyzes were purchased from Proxeon (Odense M, Denmark) and slurries were prepared in 5 탆, 100 Å pore size Magic C18 stationary phase (Michrom Bioresources, Auburn, CA) Lt; / RTI > Mobile phase A for LC separation was distilled water containing 0.1% formic acid and mobile phase B was acetonitrile containing 0.1% formic acid. The chromatographic gradient was set up with a straight line increase of 35% B at 5% B for 50 minutes, a linear increase of 60% B at 40% B for 20 minutes and an 80% B linear increase at 60% B for 5 minutes. The flow rate was maintained at 300 ml per minute of splitting. Mass spectra were obtained with data-dependent acquisition with full mass scan (400-1800 m / z) after MS / MS scan. Each MS / MS scan obtained was an average of 1 microscans in LTQ. The temperature of the ion transfer tube was adjusted to 200 ° C, and the spray was 1.5.0-2.0 kV. The normalized collision energy was adjusted to 35% for MS / MS. Sequest software was used to identify the peptide sequence. To obtain reliable results, delatCn ≥ 0.1 and Rsp ≤ 4 were applied. For Xcorr, Xcorr ≥ 1.5 was applied in charge state 1+, Xcorr ≥ 2.0 in charge state 2+, and charge state 3+ in charge state 2+ Xcorr ≥ 2.5 was applied, and the peptide identification> 0.1 was used to identify the protein as the cutoff. Peptides have been variously oxidized at methionine residues and have been carboxyamidomethylation or carboxymethylation in a variety of cysteines.

Spot No. Identified protein Accession No. Cov% pI M.W Change 1/10 LD ₅₀ 1/2 LD ₅₀ One Transketolase 1729977 25 7.3 68370 ↗ ↗ 2 Chaina, New Crystal Forms Of A Mu Class Glutathione S-Transferase From Rat Liver 442967 52 8.6 25940 ↗ ↗ 3 glutathione S-transferase, mitochondrial 310077128 69 9.13 25493 ↘ ↗ 4 beta 1 globin 546056 82 7.98 15834 ↘ ↘ 5 Hemoglobin beta-2 chain (Beta-2-globin) Hemoglobin beta chain, minor-form) 122529 46 9.2 16080 ↗ ↗ 6 Similar to Hemoglobin beta-2 subunit (Beta-2-globin) (Hemoglobin beta chain, minor-form) 109459168 82 8.91 15982 ↘ ↗ 7 keratin 10 57012436 15 5.10 57681 ↘ ↘ 8 Alpha-2u globulin 204264 46 5.4 17340 ↘ ↘

Transketolase (spot 1), mu-class glutathione S-transferase from rat liver (S-transferase from rat liver, spot 2) and hemoglobin subunit beta-2 5) increased protein expression with increasing trichlorethylene concentration (FIG. 2A). Alpha-2u globulin (spot 8) and beta 1 globin (spot 4), keratin 10 (spot 7), and alpha-2u globulin (spot 8) contained 1/10 LD ₅₀ (565 mg / kg) trichlorethylene Protein expression decreased with increasing concentration. Therefore, it can be seen that the spots 1, 2, 5, 4, 7, and 8 change in a concentration-dependent manner.

On the other hand, 1/10 LD ₅₀ (565 mg / kg) in the case of mitochondrial glutathione S-transferase (mitochondrial, spot 3) and hemoglobin beta-2 subunit In the trichlorethylene-treated group, it was decreased compared to the normal group, but increased by more than 1.5 times in the trichlorethylene treated group of 1/2 LD ₅₀ (2825 mg / kg) (FIG. 2C).

Increasing transketolase in the trichlorethylene treated group is an essential element of the pentose metabolism that catalyzes the conversion of glucose to pentose in the energy metabolism PyroPhosphate Pathway. Since the transketolase decreases early in the thiamine deficiency, activity measurement of this enzyme in red blood cells is used to assess thiamine nutritional status.

The mu class of glutathione S-transferase (S-transferase from rat liver, spot 2), mitochondrial glutathione S-transferase (mitochondrial, spot 3) An enzyme that protects tissues from toxic substances in vivo by catalyzing the binding of glutathione and an electrophilic compound has been confirmed for several polymorphisms and a person having a null genotype of some glutathione S-transferase produces glutathione S-transferase protein And affect the susceptibility of various diseases. It also works well for the detoxification of endogenous compounds such as peroxidase lipids, such as metabolism of xenobiotics. Glutathione S-transferase has the ability to detoxify activated carcinogens via glutathione incorporation.

Hemoglobin beta-2 subunit (spot 6) and hemoglobin subunit beta-2 (spot 5) and beta 1 globin (spot 4) Related protein, and performs functions related to providing oxygen necessary for a living body.

Keratin 10 (keratin 10, spot 7), which is dependent on trichlorethylene concentration, is a protein belonging to the keratin group and has been shown to be involved in activating protein synthesis machinery for cell growth for regeneration. Keratin protein is a basic component of cytoskeletal fibrous polymers known as keratin intermediate filaments and has been reported to help cells resist physical damage.

Alpha-2u globulin (spot 8) is a small protein of 20 kDa that can be found in the liver, mandibular gland, lacrimal gland, foreskin, and mammary gland of rats. It is also known as the major urinary protein and belongs to the lipocalin protein group. It is complexly regulated with hormones and is expressed in a specific way depending on tissue, age, and sex. This protein is mainly expressed in the liver and is removed from the plasma through kidneys and excretion. It is reported that male rats are more likely to have bladder cancer than humans because of the high concentration of alpha-2u globulin and albumin contained in the urine.

All of the eight proteins can be used as biomarkers for trichlorethylene. Especially, transketolase (spot 1) and alpha-2u globulin (spot 8) are useful as protein markers of trichlorethylene.

result

A total of 1170 spots were expressed in the normal group and 1176 in the case of trichlorethylene treated with 1/10 LD ₅₀ (565 mg / kg). In the case of trichlorethylene, / 2 LD ₅₀ (2825 mg / kg) treated with trichlorethylene showed 1432 spots. Among them, there were 8 protein spots showing a difference in expression between the normal group and the trichlorethylene-treated group (Fig. 1).

Figure 1a is a 2D gel photograph of a normal control without trichlorethylene treatment.

FIG. 1B is a photograph of a group treated with 1/10 LD ₅₀ (565 mg / kg) trichlorethylene.

Figure 1c shows the effect of the 1/2 LD ₅₀ (2825 mg / kg) This is a photograph of the trichlorethylene-treated group.

FIGS. 2A, 2B and 2C are photographs showing changes in protein expression of each spot by trichlorethylene. FIG.

Example 1: 1/10 LD ₅₀ (565 mg / kg) trichlorethylene treatment group,

Example 2: 1/2 LD ₅₀ (2825 mg / kg) Trichlorethylene treated group.

Claims (4)

A diagnostic kit for trichlorethylene poisoning comprising an antibody that specifically binds to a biomarker protein or an immunogenic fragment thereof comprising Transketolase; and keratin 10. The method according to claim 1, Mu class derived from rat rat glutathione S-transferase (mu class glutathione S-transferase from rat liver); Mitochondrial glutathione S-transferase (mitochondrial); Beta 1 globin; Hemoglobin beta-2 subunit; And Alpha-2u globulin; Wherein the antibody specifically binds to at least one biomarker protein or an immunogenic fragment thereof selected from the group consisting of: &lt; RTI ID = 0.0 &gt; The method according to claim 1, Wherein the kit is a kit for ELISA (Enzyme linked immunosorbent assay). . a) providing a biological sample of one of urine, blood, serum and plasma; b) contacting the biological sample with a diagnostic kit comprising an antibody that specifically binds the biomarker protein of claim 1 or 2; c) quantitatively detecting the antigen-antibody complex; And and d) comparing the detection result of step c) with a normal control group.

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