KR20210146667A - Biomarker for identification of exposure to benzene and use thereof - Google Patents

Abstract

The present invention relates to a biomarker composition for checking whether or not benzene is exposed, and a composition for checking whether or not benzene is exposed and a method for checking whether or not benzene is exposed using the same. By detecting a new biomarker checked as a metabolite of benzene from the blood of a subject suspected of being exposed to benzene, the biomarker composition for checking whether or not benzene is exposed and the composition for checking whether or not benzene, comprising an agent that detects the same, is exposed can be usefully used to determine whether or not benzene is exposed.

Description

Biomarker for identification of exposure to benzene and use thereof

The present invention relates to a biomarker composition for determining whether exposure to benzene, a composition for determining whether exposure to benzene using the same, and a method for determining whether exposure to benzene is present.

Benzene (benzene) is a colorless substance with a specific odor, very volatile, and is widely used in industry because it is used as a precursor for various materials (Schnatter et al., 1996). Benzene can be exposed in small amounts in daily life, both when working in industries that use benzene and when not. Common routes of exposure include automobile fumes and cigarette smoke, as well as small amounts of grass, paints and gasoline (Turteltaub & Mani, 2003). Benzene is highly volatile and can be inhaled. Inhaled benzene is rapidly absorbed because it passes through the lungs, and exposure to benzene causes vomiting, similar to inhaling alcohol. In particular, long-term exposure can cause hematologic cancer and hallucinations, and a recent study found that insulin expression levels could be suppressed (Amin et al., 2018).

Exposure to benzene can be determined by measuring benzene directly or by measuring its metabolites. Benzene is measured in the breath or blood, measured in amounts lower than the body's benzene level, and exposure is very inaccurate because benzene is rapidly metabolized and disappears from the blood. Another method is to detect urinary metabolites of benzene, such as muconic acid and S-penylmercapturic acid. However, phenol can be measured only when exposed to at least 10 ppm of benzene, and phenol exposed from other routes such as smoking can be detected, making the test inaccurate. In addition, the measurement of muco-acid is cumbersome to calibrate with creatine present in the urine. Therefore, the present situation is dependent on the confirmation method of whether or not benzene exposure by measuring muconic acid or S-phenyl mercapturic acid in urine.

Accordingly, the present inventors completed the present invention by identifying novel biomarkers present in the blood of the exposed group exposed to benzene compared to the normal group not exposed to benzene as a result of research to overcome the problems of the prior art.

Korean Patent Publication No. 10-2004-0012511

An object of the present invention is to provide a biomarker composition for determining whether exposure to benzene, including the compound represented by Formula 1, is provided.

In addition, an object of the present invention is to provide a composition for checking whether exposure to benzene comprising the composition.

Another object of the present invention is to provide a method for determining whether benzene is exposed, comprising detecting a compound represented by Formula 1 from blood of a subject.

One aspect of the present invention provides a biomarker composition for determining whether exposure to benzene comprising a compound represented by the following formula (1).

When exposed to a high concentration of benzene (52-62 ppm) for 4 hours, about 30% remains in the body, and 16% of it is released into the air as unmetabolized form. Benzene is mainly metabolized in the liver, with some metabolizing in the bone marrow. Benzene has a biological half-life of 24 hours in tissues with high fat content and is metabolized and excreted in the urine. The main metabolites of benzene include phenol, catechol, hydroquinone, phenyl mercapturic acid, trans muco acid, and hydroxyquinone.

As a result of analyzing the blood of mice exposed to benzene using a mass spectrometer, the present inventors confirmed that phenylsulfate represented by Formula 1 as a metabolite of benzene is a biomarker for confirming whether or not new benzene has been exposed. The compound of Formula 1 may be detected in blood by exposure to benzene.

The 'biomarker' of the present invention is generally a detectable substance in a biological sample, and organic or inorganic biomolecules such as polypeptides, proteins, nucleic acids, genes, lipids, glycolipids, glycoproteins, sugars, metabolites, etc. include all of By using these biomarkers, it is possible to objectively measure the normal or pathological state of an organism, the degree of response to a drug, and the like.

The 'biomarker composition' of the present invention may include an antibody, substrate, ligand protein, peptide, nucleic acid, chemical substance, polymer, or cofactor, etc. capable of specifically binding to a biomarker and measuring its expression level. can

In addition, another aspect of the present invention provides a composition for determining whether exposure to benzene comprising an agent for detecting the compound represented by the formula (1).

The agent may include a molecule that specifically binds to the compound of Formula 1. The molecule may be a monoclonal antibody, polyclonal antibody, substrate, ligand, protein, peptide, nucleic acid, chemical, polymer, cofactor, etc., specifically, a monoclonal recognizing compound of Formula 1 or polyclonal antibodies; specific proteins, peptides, nucleic acids, polymers or chemicals that interact with the compound of Formula 1; It may be a chemical substance or compound capable of undergoing a chemical reaction with the compound of Formula 1 to be converted into a specific chemical substance.

Such a composition for checking whether exposure to benzene may be provided as a kit.

The kit may include, in addition to molecules specifically binding to the compound of Formula 1, measurement reagents such as antibodies, proteins, peptides, nucleic acids, polymers or chemicals capable of modification (modification) nanoparticles, fluorescent dyes, etc. can

The kit may be prepared using an immunochemical reaction method, a direct interaction method, a method by a chemical reaction by adding a chemical, or the like. For example, an ELISA plate using an antibody and a flow-through device, an antibody, a chemical, a protein, or a microarray prepared by attaching to the bottom using a material having affinity for a protein or peptide conformal marker Multiple devices that can be manufactured and measured in a shape; It can be prepared in the form of a strip that can be measured through a chemical reaction with each marker or in the form of a chip coated with a molecule specifically binding to the compound of Formula 1, or the like.

When benzene exposure is confirmed using such a kit, a molecule specifically binding to the compound of Formula 1 may be brought into contact with blood collected from a subject suspected of benzene exposure. In this case, the blood may be whole blood or a state separated into plasma. By using such a kit, it is possible to check whether or not benzene has been exposed more quickly and conveniently.

In addition, another aspect of the present invention provides a method for determining whether benzene exposure, comprising the step of detecting the compound represented by Formula 1 from the blood of a subject.

The subject may be a mammal including a human.

The detection may be performed by mass spectrometry. In this case, the available mass spectrometry equipment can be used without limitation a mass spectrometer known in the art, for example, liquid chromatography-mass spectrometry (LC-MS), LC-MS/MS (liquid chromatography) -Mass Spectrometry/Mass Spectrometry), matrix-assisted laser desorption ionization (MALDI-MS), or a mass spectrometer using direct infusion.

After the step, when the compound of Formula 1 is detected in the blood or the concentration of the compound of Formula 1 is lower than that of a normal person not exposed to benzene, it can be determined that the subject has been exposed to benzene.

The biomarker composition for confirming exposure to benzene according to the present invention and the composition for checking whether or not benzene exposure comprising an agent for detecting the same according to the present invention is benzene by detecting a new biomarker identified as a metabolite of benzene from the blood of a subject suspected of benzene exposure. It can be usefully used to determine whether exposure of

1 is a schematic diagram of the metabolic pathway of benzene.
Figure 2 shows the results of profiling of metabolites in plasma: Score plots of (A) PCA model or (B) PLS-DA were applied with information on benzene exposure values between acute samples (black) and delayed samples (grey) ( ◆ Control, ▲ 100 ppm, ▼ 500 ppm, ● QC). (C) S-plot was confirmed by distinguishing acute samples in OPLS-DA from control group and benzene exposure group. (D) Each candidate substance (M47, M254) and the m/z value of the proposed substance through significance check for the database.
3 is a chromatogram for (A) a 500 ppm acute exposure sample of benzene and (B) standard 4-phenylsulfonic acid.
4 is an LC-MS/MS analysis result of (a) a 500 ppm acute exposure sample of benzene, (b) a phenyl sulfate sample of a benzene metabolite, and (c) a phenol sulfate metabolite sample: (A) is a SRM mode It is a chromatogram, and (B) is the measurement of ions produced in each sample ([MH]-, m/z 172.9914).

Hereinafter, the present invention will be described in more detail. However, these descriptions are provided for illustrative purposes only to help the understanding of the present invention, and the scope of the present invention is not limited by these illustrative descriptions.

1. Materials and Methods

1-1. reagent

Solvents water, methanol, ethyl ether and dichloromethane for mass spectrometry used in liquid chromatography (LC) were obtained from Thermo fisher scientific (USA), chloroform, hexene, ethyl ether, MTBE (methyl tert-butyl ether) and acetic acid for analysis were obtained from Sigma-Aldrich (USA). NADP ⁺ (β-nicotinamide adenine dinucleotide phosphate), MgCl ₂ , KH ₂ PO ₄ (potassium phosphate), G6P (D-glucose-6-phosphate) and G6PDH (glucose-6-phosphate dehydrogenase) are from Sigma-Aldrich (USA) was obtained from 4-phenylsulfonic acid, benzene and phenol were obtained from Tokyo Chemical Industry (Japan). The pooled rat liver S9 fraction was purchased from Corning® (USA).

1-2. animal testing

Five-week-old male C57BL/6 mice were obtained from Orient Bio Inc, Korea and bred under conditions of a temperature of 23° C., a humidity of 45-50%, and a light/dark cycle of 12/12 hours. Pellet feed and drinking water were provided ad libitum. The experimental group was randomly divided into a control group (n=6-8) and an acute or delayed benzene exposure group (100 and 500 ppm, n=6-8, respectively). The acute benzene exposure group was exposed to 100 ppm or 500 ppm of benzene for 30 minutes, and the delayed benzene exposure group had a recovery period of 2 weeks after exposure. All experiments were approved by the CHA University Animal Care and Use Committee.

1-3. Metabolite profiling and identification

(1) blood collection

Mice were exposed to 100 ppm or 500 ppm of benzene for 30 minutes. Thereafter, the mice were anesthetized and dissected, and blood collected from the abdominal aorta was used as a sample for the acute benzene exposure group. In addition, blood collected after a recovery period of 2 weeks after exposure to benzene was used as a sample of the delayed benzene exposure group. The collected blood was put in a tube containing heparin (1000 unit, 7.5 mL), shaken, and then centrifuged at 3,000 rcf for 20 minutes to collect plasma.

To extract metabolites from plasma, 50 uL of plasma was precipitated with 100 uL of acetonitrile. After centrifugation at 15,000 g for 10 minutes at 4 °C, the separated supernatant was transferred to a 1.5 mL tube. A QC (quality control) sample was prepared by mixing 10 uL of each centrifuged supernatant of each plasma, and was used to verify the stability and reproducibility of the analyzer.

(2) LC-MS/MS analysis

Plasma samples were analyzed using a mass spectrometer (Thermofisher scientific Q Exactive™ Focus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer) under the conditions shown in Table 1 below.

system Thermo Scientific™ Vanquish™ Duo UHPLC System column Kinetex C18 column (100 Х 2.1 mm id, 2.6 um particle size)
(Phenomenex, USA) mobile phase A 0.1% formic acid in water mobile phase B 0.1% formic acid in acetonitrile gradient elution
(gradient elution) hours (minutes) Condition (mobile phase B) 0-1 5% 1-8 30% 8-13 70% 14-16 95% 17-20 5% flux 0.2 mL/min sample injection volume 5 uL capillary temperature 320℃ spray pressure 3500 kV sheath gas He, flow 35 arb reserve gas He, flow rate 12 arb s-lens RF level 50 full scan resolution 70000; AGC target, 5.0X10 ⁴ maximum IT, 100 ms ddMS ² resolution 17500;
collision energy, stepped NCE 20, 40, 60 eV, maximum IT, 100 ms

(3) data processing and multivariate statistical analysis

To identify metabolites in plasma, the analysis data were processed with Compound Discoverer 2.1 (CD, Thermo-Fisher Scientific) and metabolomics technique workflow was used. At the same time, metabolites in plasma were quantified using databases such as m/z cloud, HMDB, BioCyc, and CHEBI.

1-4. Identification of candidate biomarkers for benzene exposure in plasma

(1) In vitro of benzene and phenol by S9 fraction of rat liver ( in vitro ) metabolism

To determine whether the candidate compound of the exposure marker was phenylsulfate, benzene and phenol were metabolized in the rat liver S9 fraction (RLS9 fraction), respectively. Benzene 10 mM, RLS9 fraction 1 mg/mL, 0.1 M PPB (potassium phosphate buffer) (pH 7.4), NADPH production system (3.3 mM G6P, 1.3 mM β-NADP ⁺ , 3.3 mM MgCl ₂ and G6PDH 500 unit/mL ) and PAPS 2.5 mg/mL were mixed to adjust the final volume to 100 uL and incubated at 37°C for 2 hours with stirring. The reaction was terminated by adding 50 uL of ice-cold ACN to the reaction mixture, followed by centrifugation at 4° C., 19,000 g for 10 minutes. 100 uL of the supernatant was used for LC-MS/MS analysis for metabolite analysis. In the case of phenol, it was cultured only with the coenzyme PAPS to analyze the sulfation metabolism, and the other conditions were the same as for benzene. Since benzene and phenol are very volatile solvents, they were cultured in GC vials.

(2) LC-MS/MS analysis

Samples were analyzed using a triple quadrupole mass spectrometer (TQMS) (Shimazu 8060 MS) under the conditions shown in Table 2 below.

system Shimadzu HPLC system column Kinetex C18 column (100 Х 2.1 mm id, 2.6 um particle size)
(Phenomenex, USA) mobile phase A 0.1% formic acid in water mobile phase B 0.1% formic acid in acetonitrile gradient elution hours (minutes) Condition (mobile phase B) 0-1 5% 4.5 30% 4.5-8 5% 25 30% flux 0.2 mL/min sample injection volume 1 uL Desolvation temperature 250℃ heating block temperature 400℃ spray pressure 4 kV dry gas N ₂ , flow rate 10 L/min collision gas Argon, pressure 230 kPa Nebulizing gas N ₂ , flow rate 3 L/min detection voltage 1.66 kV SRM
(selected reaction monitoring) phenyl sulfate
(172.9>93.0)

2. Results

2-1. Metabolite profiling

A total of 518 molecules were identified in metabolite profiling analysis, of which 268 were identified by databases such as HMDB and mzCloud. A VIP cutoff (VIP > 0.7) was performed to find only meaningful data among the 268 presented. As a result, it was confirmed that QC formed a cluster in the PCA score plot, and the benzene 500 ppm acute exposure group was grouped (FIG. 2A). In the score plot of the PLS-DA model, which confirmed the grouping once again by providing information about the sample, the benzene 500 ppm acute exposure group showed a difference from the control group. Both the benzene 100 ppm acute exposure group and the benzene 100 ppm or 500 ppm delayed exposure group were found to be similar to the control group ( FIG. 2B ). Then, when the OPLS-DA model was applied using samples from the acute benzene exposure group and the control group not exposed to benzene, the S-plot showed two candidate exposure markers for benzene exposure (FIG. 2C). . Candidate M47 showed significant differences in both 100 ppm and 500 ppm acute exposure groups of benzene compared to the control group. Candidate M254 showed a significant difference only in the 500 ppm acute exposure group of benzene compared to the control group (Fig. 2D). Databases such as HMDB and mzCloud suggested that candidates M47 and M254 were LPC 22: 6, 4-phenylsulfonic acid, respectively. Then, an attempt was made to identify M254, which is expected to be a metabolite of benzene.

2-2. Identification of candidate exposure markers in plasma

Sample analysis results were compared to standard 4-phenolsulfonic acid predicted in the database. As a result, it was confirmed that the retention time of the standard 4-phenolsulfonic acid was different from the retention time of the candidate M254 ( FIG. 3 ).

Then, candidate M254 and phenyl sulfate were compared. Since phenylsulfate was difficult to obtain as a standard product, an attempt was made to metabolize benzene or phenol using RLS9. As a result, it was confirmed that the retention times of the benzene metabolite and the phenol metabolite were the same as the retention times of the candidate M254, and the spectrum was also the same as that of the candidate M254 ( FIG. 4 ).

As a result, it was confirmed through this experiment that phenyl sulfate is a detectable metabolite of benzene from blood.

So far, the present invention has been looked at with respect to preferred embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (8)

A biomarker composition for determining whether exposure to benzene, comprising a compound represented by the following formula (1).
[Formula 1]

The method according to claim 1,
The compound of Formula 1 is a composition that is detected in blood by exposure to benzene.
A composition for determining whether exposure to benzene, comprising an agent for detecting a compound represented by the following formula (1).
[Formula 1]

4. The method according to claim 3,
The agent is one selected from the group consisting of monoclonal antibodies, polyclonal antibodies, substrates, ligands, proteins, peptides, nucleic acids, chemicals, polymers, and cofactors that specifically bind to the compound of Formula 1 The composition that is more than one.
A method for determining whether benzene is exposed, comprising the step of detecting a compound represented by Formula 1 from the blood of a subject.
[Claim 1]

6. The method of claim 5,
The method of claim 1, wherein the subject is a mammal including a human.
6. The method of claim 5,
wherein said detection is performed by mass spectrometry.
6. The method of claim 5,
After the step, when the compound of Formula 1 is detected in the blood, the method further comprising the step of determining that the subject has been exposed to benzene.

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