KR20110064996A - Proteomics biomarker for identification of exposure to phthalate compounds as environmental toxic materials - Google Patents

Abstract

PURPOSE: A proteomics biomarker for detecting phthalate exposure is provided to prevent or treat diseases occurrence by phthalate exposure. CONSTITUTION: A biomarker for detecting phthalate exposure contains Cystatin C, Rho-GDIα, Retinol Binding Protein, Gelsolin, DEK, Raf Kinase Inhibitor Protein, Triose Phosphate Inhibitor, Cofilin-1, or Haptoglobi. The phthalate is a compound of chemical formula 1. In chemical formula 1, R1 and R2 is independently C6-12 aryl-substituted or non-substituted alkyl of 1-24 carbon atoms or aryl of 6-12 carbon atoms. A protein array for detecting phthalate exposure contains the proteomics biomarker. The proteomics biomarker is isotope, Cy3, Cy5, FITC(poly L-lysine-fluorescein isothiocyanate), RITC(rhodamine-B-isothiocyanate), or rhodamine.

Description

Proteomics biomarker for identification of exposure to phthalate compounds as environmental toxic materials}

The present invention relates to a protein biomarker for confirming the exposure to environmentally harmful substances phthalates, protein proteins that specifically increase or decrease the expression of protein through proteome analysis in hepatocytes or blood exposed to di-2-ethylhexyl phthalate The present invention relates to protein biomarkers for identifying exposure to phthalates, as well as quantitative assessment of phthalate exposure by identifying indicators, as well as preventing or treating diseases caused by phthalate exposure.

Recently, the public's interest in environmental problems is increasing, and there are many controversies related to health problems at low levels of exposure to environmental hazardous substances, and until now, risk assessment methods using statistical modeling It depends on the situation.

Key topics in next-generation toxicity assessments based on the molecular-level biomarkers currently being pursued worldwide are toxic genomics based on the genomics and metabolomics of proteomics and transcriptomics. Toxicogenomics studies.

Toxic proteomics research can quantitatively evaluate human health risks by identifying specific biomarkers of environmentally hazardous substances, and thus can take measures to prevent the occurrence of future diseases due to environmentally harmful substances.

The development of exposure and risk assessment techniques for environmentally harmful substances through the discovery of protein markers based on toxic proteomics and the development of diagnostic kits has established systematization and standardization of research to advance the next generation of environmentally harmful substances using molecular protein biomarkers at the molecular level. Exposure and risk assessment techniques can be established and used as strategic skills at the national level.

However, no protein index has been reported to confirm the exposure of phthalates.

An object of the present invention is to provide a protein biomarker that can determine whether the environmental toxic substances phthalates exposed in vivo.

In order to achieve the above object, the present invention is a phthalate containing at least one selected from the group consisting of Cystatin C, Rho-GDIα, Retinol Binding Protein, Gelsolin, DEK, Raf Kinase Inhibitor Protein, Triose Phosphate Inhibitor, Cofilin-1 and Haptoglobin It provides a protein biomarker to confirm exposure.

The present invention also provides a protein array for confirming exposure of a phthalate comprising a protein biomarker according to the present invention.

The present invention also provides a kit for confirming exposure of a phthalate comprising the protein array of the present invention.

The present invention also provides a method for identifying phthalate exposure using the protein biomarker according to the present invention.

The present invention can quantitatively detect whether or not phthalates, which are environmentally harmful substances, are exposed in vivo, and furthermore, can be utilized as important information in the prevention or treatment of diseases related to environmentally hazardous substances.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

The present invention provides a protein biomarker for phthalate exposure including at least one selected from the group consisting of Cystatin C, Rho-GDIα, Retinol Binding Protein, Gelsolin, DEK, Raf Kinase Inhibitor Protein, Triose Phosphate Inhibitor, Cofilin-1, and Haptoglobin. (biomarker).

Protein biomarker for confirming whether or not phthalate exposure of the present invention is characterized in that the protein showing a difference in expression frequency in human somatic cells, blood or the like exposed to phthalate compounds. The difference in the expression frequency of the protein can be understood as an increase or decrease in expression in a living body exposed to phthalate compounds.

The phthalate compound may be a compound represented by Formula 1 below:

[Formula 1]

Where

R ₁ and R ₂ each independently represent an alkyl having 1 to 24 carbon atoms or an aryl having 6 to 12 carbon atoms unsubstituted or substituted with aryl having 6 to 12 carbon atoms.

The terms used in the definition of the substituents of the compound of formula 1 of the present invention are as follows.

"Alkyl" refers to a straight or branched chain saturated hydrocarbon having 1 to 24 carbon atoms unless otherwise specified. Examples of C _1-24 alkyl groups are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, isopropyl, isobutyl, sec-butyl and tert-butyl, isopentyl, neopentyl, isohexyl , Isoheptyl, isooctyl, isononyl and isodecyl, but are not limited to these

"Aryl" refers to a 6-12 membered aromatic cyclic compound unless otherwise stated. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl and anthracenyl.

Specifically, the compound of Formula 1,

R ₁ and R ₂ may be each independently alkyl having 1 to 12 carbon atoms.

Most specifically, the compound of Formula 1 may be di-2-ethylhexyl phthalate.

The protein biomarker is a protein that increases or decreases expression in vivo when exposed to di-2-ethylhexyl phthalate,

Specifically, Rho-GDIα, Gelsolin, Raf Kinase Inhibitor Protein, Triose Phosphate Inhibitor, or Cofilin-1 is a protein whose expression is increased upon phthalate exposure, most specifically, the Rho-GDIα, Gelsolin, Raf Kinase Inhibitor Protein and Triose Phosphate Inhibitor is a protein that increases expression depending on phthalate exposure concentration and Cofilin-1 is a protein that increases expression depending on phthalate exposure time.

The Rho-GDIα is not particularly limited as long as it is a known human-derived Rho-GDIα amino acid sequence, and is registered in http://www.uniprot.org/ . The amino acid sequence described in IPI00003815 can be used.

Gelsolin is not particularly limited as long as it is a known human-derived Gelsolin amino acid sequence, accession no. Registered at http://www.uniprot.org/ . The amino acid sequence described in IPI00026314 can be used.

The Raf Kinase Inhibitor Protein is not particularly limited as long as it is a known human-derived Raf Kinase Inhibitor Protein amino acid sequence, and is registered in http://www.uniprot.org/ . The amino acid sequence described in IPI00219446 can be used.

The Triose Phosphate Inhibitor is not particularly limited as long as it is a known human-derived Triose Phosphate Inhibitor amino acid sequence, but accession no. Registered at http://www.uniprot.org/ . The amino acid sequence described in IPI00451401 can be used.

The Cofilin-1 is not particularly limited as long as it is a known human-derived Cofilin-1 amino acid sequence, but accession no. Registered at http://www.uniprot.org/ . The amino acid sequence described in IPI00012011.6 can be used.

In addition, Cystatin C, Retinol Binding Protein, DEK, or Haptoglobin is a protein whose expression is decreased upon phthalate exposure. More specifically, Cystatin C, Retinol Binding Protein, or DEK is decreased in phthalate exposure concentration. Phthalate exposure time is a protein whose expression decreases.

The Cystatin C is not particularly limited as long as it is a known human-derived Cystatin C amino acid sequence, and is registered in http://www.uniprot.org/ . The amino acid sequence described in IPI00032293 can be used.

The Retinol Binding Protein is not particularly limited as long as it is a known human-derived Retinol Binding Protein amino acid sequence, accession no. Registered at http://www.uniprot.org/ . The amino acid sequence described in IPI00022420 can be used.

The DEK is not particularly limited as long as it is a known human-derived DEK amino acid sequence, but accession no. Registered at http://www.uniprot.org/ . The amino acid sequence described in IPI00020021.3 can be used.

Haptoglobin is not particularly limited as long as it is a known human-derived Haptoglobin amino acid sequence, accession no. Registered at http://www.uniprot.org/ . The amino acid sequence described in IPI00477597.1 can be used.

The present invention also relates to a protein array for confirming exposure of a phthalate comprising the protein biomarker of the present invention.

The protein biomarker included in the protein array for phthalate exposure check of the present invention is understood to be an absolute amount of a comparative substance that is used in comparison to similar or identical analogs to quantify the amount of protein present in the sample. Can be.

The protein of the sample may be obtained from somatic cells or blood of the subject, but is not particularly limited thereto.

The somatic cells may be hepatocytes, but are not particularly limited thereto.

As used herein, an “array” is an elemental arrangement (such as a polypeptide) present on a solid support and / or in a container arrangement. Arrays are most often thought of as physical elements having a specific space-physical relationship, and the present invention may use "logical" arrays that do not have linear spatial organization. For example, a computer system may be used to track the location of one or several of those components that are present on or among physically different components. The computer system creates a logical array by providing a "index" table of the physical location of the array members. Thus, the moving component may also be part of the logical array, as long as the members of the array are specified and located. This is appropriate when the array of the present invention is present in a flow microscale system or when present in one or more microtiter trays.

Certain array formats are sometimes referred to as "chips" or "biochips." The array may include, for example, a low density of 2 to about 10 addressable locations, a medium density of about 100 or more locations, or a number of high density, for example 1000 or more. Typically, chip array formats are geometrically consistent shapes that allow for easy assembly, handling, placement, stacking, reagent introduction, detection, and storage. However, it may be irregular. As one typical format, the array is structured in a landscape format, with regular gaps between each location of the set of members on the array. Alternatively, the locations can be bundled, blended or homogenized for even processing or sampling. The array may include a plurality of addressing locations, each structured such that high throughput handling, automatic delivery, masking, or sampling of reagents is spatially addressed. The array can facilitate detection or quantification by specific means, including scanning by laser irradiation, confocal or deflected light collection, CCD detection, and chemiluminescence, and is not limited to the above examples. An "array" format as described herein may be used in conjunction with arrays (ie, arrays of multiple chips), microchips, microarrays, microarrays assembled on a single chip, arrays of biomolecules attached to microwell plates, or such systems. Other suitable formats for use include, but are not limited to these.

Phthalate exposure protein array of the present invention can be produced by methods known to those skilled in the art.

Specifically, in order to immobilize the detected protein biomarker as a probe molecule (probe) on the substrate of the protein array, a micropipetting method or a pin type using a piezoelectric method It is preferable to use a method using a spotter or the like, but is not limited thereto. The substrate of the protein array is preferably coated with one active group selected from amino-silane, poly-L-lysine, aldehyde, and the like, but is not limited thereto. . In addition, the substrate may be a slide glass, plastic, metal, silicon, nylon film, or nitrocellulose film, but is not particularly limited thereto.

The protein biomarker for use as the probe molecule may be labeled with isotopes or fluorescent materials such as Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate (RITC), or rhodamine. Can be.

The present invention also relates to a kit for confirming exposure of a phthalate comprising the protein array of the present invention.

The phthalate exposure check kit of the present invention is characterized by being able to confirm the exposure to the phthalate compounds by quantitatively analyzing the protein of the sample including one or more protein biomarkers.

Kits of the present invention may be incorporated into a combined packaging material (material for packaging an array), instructions (e.g., instructions for using the array to detect one or more reagents or samples interacting with the array), control reagents (applied to the array) Known kit's active agents), samples, and the like, together with additional kit features.

The present invention also relates to a method for identifying phthalate exposure using the protein biomarker of the present invention.

Phthalate exposure check method of the present invention is

Contacting the subject's protein sample with a labeled protein biomarker; And

It is preferable to include the step of confirming the expression level of the protein biomarker according to the contact compared with the control.

In the method for confirming exposure of phthalate of the present invention, the first step is a step of contacting a protein sample of a subject with a labeled protein biomarker.

The protein sample of the subject may be isolated from somatic cells or blood of the subject, but is not particularly limited thereto.

The somatic cells may be hepatocytes, but are not particularly limited thereto.

The blood may be specifically plasma, but is not particularly limited thereto.

The method of separating proteins from the somatic cells or blood of the subject can use a conventional protein extraction method, and is not particularly limited.

The contact between the protein sample and the labeled protein biomarker isotope; Or a microtiter comprising a protein biomarker of the present invention labeled with at least one fluorescent substance such as Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate (RITC), or rhodamine It can be made in the plate.

In the method for confirming the exposure of the phthalate of the present invention, the second step is a step of confirming the expression level of the protein biomarker according to the contact with the control.

The method for confirming the expression level of the protein biomarker can be used by methods known to those skilled in the art, and is not particularly limited.

In one embodiment, the ionic strength obtained with the appropriate protein biomarker can be used to calculate the concentration of the target protein. Protein biomarkers are identified using a single characteristic ion pair (or several ion pairs), and the ionic strength obtained is related to the known concentration of the protein biomarker, allowing the quantification of the corresponding target protein. Target proteins are known in advance and can be pre-tinned. Thus, the detected and quantified protein is already annotated, allowing fast and direct interpretation.

Mass measurement of the target protein may use a mass spectrometer capable of MSMS analysis to distinguish ionic species, but is not particularly limited thereto.

From the above results, the increase in ionic strength by quantitative analysis of the protein can confirm whether the phthalate compounds are exposed in vivo.

According to one embodiment of the invention, if an increase or decrease in ionic strength of Cystatin C, Rho-GDIα, Retinol Binding Protein, Gelsolin, DEK, Raf Kinase Inhibitor Protein, Triose Phosphate Inhibitor, Cofilin-1, or Haptoglobin is observed in a sample And di-2-ethylhexyl phthalate.

Hereinafter, the present invention will be described in more detail through examples according to the present invention and comparative examples not according to the present invention, but the scope of the present invention is not limited to the examples given below.

Example 1 Cell and Genetic Toxicity Studies on Environmentally Harmful Substances in Human Hepatocyte Cell Lines

Toxicity concentrations of DEHP for genotoxicity indicators were established by measuring genotoxicity and cytotoxicity of di-2-ethylhexyl phthalate in human hepatocytes.

HepG2 cells (ATCC) were used as human hepatocytes and cultured in MEM (WelGENE Inc., Korea) medium.

Di-2-ethylhexyl phthalate exposure to human hepatocytes was performed at 37 ° C. for 24 or 48 hours in a medium containing 0 μM to 250 μM di-2-ethylhexyl phthalate (sigma) dissolved in 0.1% DMSO (sigma).

After the exposure, the culture supernatant was collected and purified from the Microcon YM3 Column (Milipore Co. MA, USA).

Genotoxicity and cytotoxicity in hepatocytes was performed using MTT and Comet assays.

Figure 1 shows the results of genotoxicity in human hepatocytes of di-2-ethylhexyl phthalate using Comet analysis, it can be seen that significantly causes genotoxicity from a concentration of 2.5μM or more.

Figure 2 shows the cytotoxicity results of di-2-ethylhexyl phthalate in human hepatocyte cell line using MTT assay, it can be seen that it causes significant cytotoxicity from the concentration of 25μM or more.

From the above results, it was found that as the concentration of di-2-ethylhexyl phthalate and the exposure time increased, genotoxicity and cytotoxicity increased, and 5, 10 μM, which is the maximum concentration that causes genotoxicity but does not cause cytotoxicity, was selected. The experiment was conducted.

Example 2 Discovery of Protein Indicators for Di-2-ethylhexyl Phthalate in Human Hepatocyte Cell Lines

For protein index detection of di-2-ethylhexyl phthalate in human hepatocytes, the protein samples were prepared using narrow range immobiline dry strips (pH 4-7, pH 6-9, 24 cm) at fixed pH. Dimensional electrophoresis was performed.

Next, the protein was separated from human hepatocytes by 2-DE analysis (12% SDS-PAGE, 34 × 45 cm, OWL), and the protein biomarker for DEHP was analyzed by silver staining followed by image analysis (Progenesis Discovery (Nonlinear)). Indicators were excavated (FIG. 3).

Next, accurate protein identification was performed through the European Bioinformatics Institute (EBI) 's IPI human database using ESI-MS / MS (LCQ Deca XP-Plus, Thermo Finnigan, USA) and Western blot.

As shown in Table 1 and Figure 4, Rho-GDIα, Gelsolin, Raf Kinase Inhibitor Protein, Triose Phosphate Inhibitor, or Cofilin-1 increased expression upon phthalate exposure, in particular, Rho-GDIα, Gelsolin, Raf Kinase Inhibitor Protein And Triose Phosphate Inhibitor increased the expression of phthalate exposure concentration and Cofilin-1 increased the expression of phthalate exposure time.

Cystatin C, Retinol Binding Protein, DEK, or Haptoglobin is a protein that is degraded upon phthalate exposure, especially Cystatin C, Retinol Binding Protein, or DEK decreases in phthalate exposure concentration-dependent manner, while Haptoglobin is phthalate exposure time dependent. Expression decreased.

Therefore, Rho-GDIα, Gelsolin, Raf Kinase Inhibitor Protein, Triose Phosphate Inhibitor, Cofilin-1, Cystatin C, Retinol Binding Protein, DEK, Haptoglobin as proteomic indicators for human exposure assessment to di-2-ethylhexyl phthalate. It was confirmed that it could be used.

Figure 1 shows the genotoxicity results of di-2-ethylhexyl phthalate in human hepatocytes using Comet assay.

Figure 2 shows the cytotoxicity results of di-2-ethylhexyl phthalate in human hepatocytes using MTT assay.

3 shows two-dimensional electrophoresis patterns of proteins of human hepatocytes exposed to di-2-ethylhexyl phthalate using various IEF strips and large gel systems.

Figure 4 shows the expression pattern of proteins of human hepatocytes exposed to di-2-ethylhexyl phthalate by Western blot analysis.

Claims (14)

Protein biomarker for phthalate exposure including at least one selected from the group consisting of Cystatin C, Rho-GDIα, Retinol Binding Protein, Gelsolin, DEK, Raf Kinase Inhibitor Protein, Triose Phosphate Inhibitor, Cofilin-1, and Haptoglobin . The method of claim 1, Phthalate is a protein biomarker for confirming exposure of phthalate, a compound represented by the following formula (1): [Formula 1]

Where R ₁ and R ₂ each independently represent an alkyl having 1 to 24 carbon atoms or an aryl having 6 to 12 carbon atoms unsubstituted or substituted with aryl having 6 to 12 carbon atoms. The method of claim 2, R ₁ and R ₂ are each independently a protein biomarker for phthalate exposure is alkyl having 1 to 12 carbon atoms. The method of claim 2, Protein biomarker for confirming whether the compound of formula 1 is phthalate exposure is di-2-ethylhexyl phthalate (di-2-ethylhexyl phthalate). The method of claim 1, Rho-GDIα, Gelsolin, Raf Kinase Inhibitor Protein, Triose Phosphate Inhibitor, or Cofilin-1 is a protein biomarker for phthalate exposure that expression is increased by phthalate exposure. The method of claim 1, Cystatin C, Retinol Binding Protein, DEK, or Haptoglobin is a protein biomarker for phthalate exposure that expression is reduced by phthalate exposure. A protein array for confirming exposure of phthalate comprising the protein biomarker according to any one of claims 1 to 6. The method of claim 7, wherein Proteomic biomarkers are exposed to isotopes or phthalates labeled with one or more phosphors selected from the group consisting of Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate (RITC), and rhodamine Identification protein array. A kit for confirming exposure of a phthalate comprising a protein array according to any one of claims 7 and 8. A method for confirming exposure of phthalate using the protein biomarker according to any one of claims 1 to 6. The method of claim 10, Contacting the subject's protein sample with a labeled protein biomarker; And Phthalate exposure check method comprising the step of confirming the expression level of the protein biomarker according to the contact compared to the control. The method of claim 11, Protein samples are isolated from somatic cells or blood of a subject to determine whether phthalate exposure. The method of claim 12, Somatic cells of the subject is a hepatocyte phthalate exposure method. The method of claim 12, The blood of the subject is a plasma phthalate exposure method.

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