US20130171655A1 - Novel biological detection method for dioxins in serum, and a diagnostic use therefor in metabolic syndrome and related conditions - Google Patents

Novel biological detection method for dioxins in serum, and a diagnostic use therefor in metabolic syndrome and related conditions Download PDF

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US20130171655A1
US20130171655A1 US13/821,779 US201113821779A US2013171655A1 US 20130171655 A1 US20130171655 A1 US 20130171655A1 US 201113821779 A US201113821779 A US 201113821779A US 2013171655 A1 US2013171655 A1 US 2013171655A1
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serum
dioxin
cell line
reporter gene
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Hong Kyu Lee
YoungMi Kim
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Industry Academic Cooperation Foundation of Kyung Hee University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5067—Liver cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C12N2015/8527—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic for producing animal models, e.g. for tests or diseases
    • C12N2015/8572—Animal models for proliferative diseases, e.g. comprising an oncogene
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders

Definitions

  • the present invention relates to a novel biological detection method for dioxin like activity in serum and a diagnostic use thereof in metabolic syndrome and related conditions.
  • POPs Persistent organic pollutants
  • pops do not exist in the nature or only very small amounts of them if they exist. They are mostly organic chemicals generated artificially for human needs or byproducts of industrialization. Dioxin and dioxin like substances are the representatives. Once POPs are come into human body, they are not easily decomposed or excreted, resulting in the accumulation in human body. Recently, the public is more interested in the situation of these chemicals in our environment, including industrial byproducts, pesticides, or drugs cause potential endocrine disruption in human and many other wild species.
  • Dioxin compounds one of the most representative POPs, are called environmental hormone or endocrine disrupter, which have been important issues world widely.
  • TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin
  • TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin
  • TCDD ubiquinone
  • dioxin is introduced in a cell, it is specifically linked to arylhydrocarbon receptor (AhR) in cytoplasm.
  • This dioxin-receptor complex is then introduced in the nucleus and the complex is bound to ARNT (AhR nuclear translocator) therein.
  • ARNT AhR nuclear translocator
  • the complex is converted into the DNA bindable complex.
  • the transformed DNA bindable complex is strongly conjugated to a specific gene and at this time such specific gene is called dioxin responsive element (DRE).
  • DRE dioxin responsive element
  • the dioxin complex is linked to DRE, the neighboring response gene P4501A1 is activated to induce the synthesis of cytochrome P4501A1 enzyme. Then, by those synthesized enzymes, dioxin toxicity is expressed.
  • TCDDEq concentration of TCDD equivalent
  • instrumental method has been widely used.
  • the method for analyzing TCDDEq up to date is exemplified by gas chromatography/mass spectrometry (GC/MS) and cell-based assay.
  • GC/MS enables the analysis of concentration of each pollutant including TCDD and the detection thereof.
  • high price has to be paid.
  • a large amount of serum sample (25 ⁇ 200 ml) is required for the analysis as well.
  • cell-based assay which is exemplified by CALUX (Chemically Activated Luciferase Expression) is a biological quantification method to detect dioxin compounds by using the cell line secreting luciferase in the presence of dioxin constructed by gene manipulation.
  • CALUX Chemical Activated Luciferase Expression
  • dioxin-like compounds can be measured and TCDD equivalency (TEQ) can also be measured.
  • TEQ TCDD equivalency
  • analysis with the said method takes long and asks a large amount of serum for the extraction or purification process using hexane from human serum samples (at least, 1 ml of serum is necessary, and generally 10 ⁇ 20 ml of sample is required).
  • the transient transfection cell line is used, inter-assay variation is very big, leaving a question in reliability. As the number of samples increases, handling speed and reliability decrease.
  • dioxin extraction step is required. For example, a sample is treated with an organic solvent to extract fat, which is passed through the activated acid silica gel column to purify the fat.
  • the conventional methods require high priced equipments for the analysis and the complicated pre-treatment step for sample purification, for which a large volume of organic solvents and other toxic reagents such as radio-labeled materials are used and longer analyzing time is taken. Therefore, it is required to develop and adapt a novel biological method realizing test and quantification with simple pre-treatment step within a short period of time with guaranteeing high detection sensitivity for saving time, effort, and costs.
  • the present inventors studied to develop a novel high throughput detection method for POPs which has improvement from the conventional detection methods for POPs and is easy and accurate.
  • a novel biological detection method which uses total serum for easy but accurate analysis of multiple samples and is characterized by accurate analysis even with a small volume of serum has been established.
  • mutant genes and inventions using the same have been accepted as targets for patent application world-widely. Therefore, the present inventors have completed this invention by confirming that DRE gene could be effectively used for the detection of dioxin compounds.
  • the present invention provides a novel biological detection method for dioxins in serum, which comprises the following steps:
  • DRE dioxin-responsive elements
  • step 2) culturing the transformed cell line prepared in step 1) after treating the cell line with the serum of a test subject;
  • the present invention also provides a method for predicting the likelihood of diabetes or metabolic syndrome, which comprises the following steps:
  • DRE dioxin-responsive elements
  • step 2) culturing the transformed cell line prepared in step 1) after treating the cell line with the serum of a test subject;
  • step 3 determining the likelihood of diabetes or metabolic syndrome when the reporter gene expression is detected in step 3).
  • the present invention also provides a method for monitoring the prognosis of diabetes or metabolic syndrome, which comprises the following steps:
  • DRE dioxin-responsive elements
  • step 2) culturing the transformed cell line prepared in step 1) after treating the cell line with the serum of a test subject with diabetes or metabolic syndrome treated;
  • step 4) judging that diabetes or metabolic syndrome of the test subject is alleviated, improved or treated when the expression level of the reporter gene detected in step 3) is lowered, compared with that of the control treated with serum of a test subject with diabetes or metabolic syndrome non-treated.
  • the present invention provides a preparation method of a foreign protein with the original form.
  • the detection method for environmental hormone of the present invention has an improvement from the conventional one that requires the step of purification of dioxins from the serum before measurement and has an advantage of saving time and effort because of using serum as a whole, which thereby enables the analysis with increased accuracy and easiness even with multiple numbers of samples.
  • This method is advantageous in biological detection of environmental hormone in serum owing to the efficiency in analysis with small amount of samples without pre-treatment with hexane.
  • this method can be used to research the correlation between specific POPs and patients' disease factors by accurately detecting whether POPs such as dioxins are present in the serum. This method can be further used effectively to predict the occurrence of disease and to determine treatability.
  • FIG. 1 is a set of diagrams illustrating the cloning of pCYP1A1-luc vector.
  • FIG. 2 is a photomicrograph showing the colonies of Hepa1C1c7 cell line expressing pCYP1A1-luc stably.
  • FIG. 3 is a graph illustrating the results of indole-3-carbinol response test with HePa1C1c7 cell line expressing pCYP1A1-luc stably.
  • FIG. 4 is a graph illustrating the results of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) response test with HePa1C1c7 cell line expressing pCYP1A1-luc stably.
  • TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin
  • FIG. 5 is a graph illustrating the distribution of dioxin fold induction values.
  • FIG. 6 is a graph illustrating the dioxin fold induction value in human serum over the body weight analyzed by linear regression analysis.
  • FIG. 7 is a graph illustrating the dioxin fold induction value in human serum over the bmi analyzed by linear regression analysis.
  • FIG. 8 is a graph illustrating the dioxin fold induction value in human serum over the we analyzed by linear regression analysis.
  • FIG. 9 is a graph illustrating the dioxin fold induction value in human serum over the sbp analyzed by linear regression analysis.
  • FIG. 10 is a graph illustrating the dioxin fold induction value in human serum over the tg analyzed by linear regression analysis.
  • FIG. 11 is a graph illustrating the dioxin fold induction value in human serum over the fbs analyzed by linear regression analysis.
  • the present invention provides a biological detection method for environmental hormone in serum by using a transformed cell line comprising a recombinant reporter gene whose expression is modified by environmental hormone.
  • the said detection method is composed of the following steps, but not always limited thereto:
  • DRE dioxin-responsive elements
  • step 2) culturing the transformed cell line prepared in step 1) after treating the cell line with the serum of a test subject;
  • step 3 determining the likelihood of diabetes or metabolic syndrome when the reporter gene expression is detected in step 3).
  • the said dioxin compounds are preferably polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzo-furans (PCDFs), polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarcons (PAHs), flavonoids, or pesticides, but not always limited thereto, and any environmental hormone that can affect transcription activity by combining with the said DRE, in addition to dioxin-like compounds, can be included.
  • PCDDs polychlorinated dibenzodioxins
  • PCDFs polychlorinated dibenzo-furans
  • PCBs polychlorinated biphenyls
  • PAHs polycyclic aromatic hydrocarcons
  • flavonoids flavonoids
  • At least one of the said dioxin-responsive elements of step 1) is included and most preferably 3 ⁇ 4 DREs are included, but not always limited thereto.
  • the promoter of step 1) is preferably selected from the group consisting of MMTV (Mouse Mammary Tumor Virus) promoter, SV40 promoter, and CMV (cytomegalovirus) promoter, and is more preferably MMTV (Mouse Mammary Tumor Virus) derived promoter with elimination of glucocorticoid response element region, but not always limited thereto.
  • MMTV Mammary Tumor Virus
  • the promoter herein can be a minimum promoter excluding enhancer. Transcription activity of a reporter gene can be determined by the enhancer or cis-acting element near the upstream of a minimum promoter. Therefore, removing enhancer means eliminating other elements that can affect transcription activity.
  • the reporter gene of step 1) is preferably selected from the group consisting of luciferase, alkaline phosphatase, chloramphenicol acetyl transferase, and ⁇ -galactosidase, and is more preferably luciferase if considering convenience and sensitivity of activity analysis, but not always limited thereto.
  • the transformed cell line herein indicates the cell line in which the reporter gene is stably expressed. That is, the reporter vector becomes stably a part of a genetic repertory in the host cell once the transformed reporter vector is introduced in the chromosome of the host cell.
  • the stable expression of the introduced gene in the transformed reporter vector can be lasted at least 30 generations.
  • the host cell of step 1) is preferably eukaryotic, mammalian cell, and more preferably mammalian tumor cell, and most preferably mouse hepatocarcinoma cell line, for example Hepa1c1c7 cell line, but not always limited thereto.
  • arylhydrocarbon receptor AhR
  • ARNT AhR nuclear translocator
  • the transformation herein can be performed by electroporation, plasmogamy, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, agitation using silicon carbide fiber, or lipofectamine mediated method, but not always limited thereto.
  • the conventional detection method for dioxin compounds requires a preprocessing step to extract dioxins through purification of fat extracted from a sample by treating an organic solvent.
  • the serum of step 2) is total serum, indicating the serum as a whole can be used.
  • the serum herein is preferably heat-inactivated for the matter of safety of serum later in the use in the transformed cell line, but not always limited thereto.
  • mouse CYP1A1 promoter comprising 4 dioxin responsive element (DRE) binding sites (5′-TNGCGTG-3′) represented by SEQ. ID. NO: 1 and a recombinant reporter gene vector comprising mouse mammary tumor virus (MMTV) long terminal repeat (LTP) (see FIG. 1 ) were constructed.
  • DRE dioxin responsive element
  • MMTV mouse mammary tumor virus
  • LTP long terminal repeat
  • luciferase activity was significantly increased when TCDD was treated thereto at the concentration of 10 ⁇ 1000 pM (see FIG. 4 ). Therefore, the cell line stably expressing the recombinant reporter gene was confirmed to react to TCDD and to activate luciferase effectively.
  • the present inventors detected dioxins in human serum sample by using the constructed cell line and then analyzed the correlation between dioxin content and physical variables.
  • serum samples were obtained from 97 people, followed by heat inactivation at 65° C. for 30 minutes for the safety of cell culture.
  • the heat-inactivation herein indicates to eliminate biological activity of serum by heating.
  • Serum contains not only cell growth factors and serum proteins but also complements which are the factors responsible for humoral immunity. Many complements demonstrate cytotoxicity by recognizing cultivated cells, so that when serum is added as a whole to the medium, cell growth is suppressed or worse the cell line might be killed. So, for the safety of cell culture, serum needs to be heat-inactivated.
  • dioxin fold induction value was higher in drinkers/smokers than in non-drinkers/non-smokers.
  • IGR impaired glucose regulation
  • IGF impaired glucose tolerance
  • IGF impaired fasting glucose
  • DM diabetes mellitus
  • GNT normal glucose tolerance
  • the method of the present invention was compared with the conventional ones in the aspect of technical matters and effect, it was confirmed as follows: the conventional methods require a preprocessing step to extract dioxins from serum by using an organic solvent like hexane in order to use CALUX analysis system effectively as shown in Table 1.
  • the present invention is advantageous in saving time, costs, and labor by analyzing serum simply heat-inactivated.
  • the system of the present invention is advantageous. That is, even with approximately 1/1000 of serum required for the analysis by the conventional method, the system of the invention can analyze serum with high efficiency with similar sensitivity to that of the previous method using a large amount of serum.
  • the dioxin content in serum detected by the detection method for dioxins of the present invention was significantly correlated with various physical variables.
  • the method of the present invention is advantageous in easy and fast analysis of multiple samples with saving time and labor by using serum as a whole and also advantageous in accurate analysis even with a small amount of each sample.
  • the transformed cell line harboring a recombinant reporter gene whose expression is regulated by environmental hormone and total serum can be effectively used for the biological detection of environmental hormone in serum with this method.
  • the present invention also provides a method for predicting the likelihood of diabetes or metabolic syndrome by using a transformed cell line harboring a recombinant reporter gene whose expression is regulated by dioxin compounds.
  • the method for predicting is preferably composed of the following steps, but not always limited thereto:
  • DRE dioxin-responsive elements
  • step 2) culturing the transformed cell line prepared in step 1) after treating the cell line with the serum of a test subject;
  • step 3 determining the likelihood of diabetes or metabolic syndrome when the reporter gene expression is detected in step 3).
  • the present invention also provides a method for monitoring the prognosis of diabetes or metabolic syndrome by using a transformed cell line harboring a recombinant reporter gene whose expression is regulated by dioxin compounds and total serum.
  • the method for monitoring is preferably composed of the following steps, but not always limited thereto:
  • DRE dioxin-responsive elements
  • step 2) culturing the transformed cell line prepared in step 1) after treating the cell line with the serum of a test subject with diabetes or metabolic syndrome treated;
  • step 4) judging that diabetes or metabolic syndrome of the test subject is alleviated, improved or treated when the expression level of the reporter gene detected in step 3) is lowered, compared with that of the control treated with serum of a test subject with diabetes or metabolic syndrome non-treated.
  • At least one of the said dioxin-responsive elements of step 1) is included and most preferably 3 ⁇ 4 DREs are included, but not always limited thereto.
  • the promoter of step 1) is preferably selected from the group consisting of MMTV (Mouse Mammary Tumor Virus) promoter, SV40 promoter, and CMV (cytomegalovirus) promoter, and is more preferably MMTV (Mouse Mammary Tumor Virus) derived promoter, but not always limited thereto.
  • the reporter gene of step 1) is preferably selected from the group consisting of luciferase, alkaline phosphatase, chloramphenicol acetyl transferase, and p-galactosidase, and is more preferably luciferase if considering convenience and sensitivity of activity analysis, but not always limited thereto.
  • the host cell of step 1) is preferably eukaryotic, mammalian cell, and more preferably mammalian tumor cell, and most preferably mouse hepatocarcinoma cell line, for example Hepa1c1c7 cell line, but not always limited thereto. Any cell line that can express ARNT and AhR endogenously can be used as a host cell line herein.
  • the transformation herein can be performed by electroporation, plasmogamy, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, agitation using silicon carbide fiber, or lipofectamine mediated method, but not always limited thereto.
  • the serum of step 2) is total serum that can be used as a whole without a preprocessing step for purifying dioxins from the serum.
  • the serum is preferably heat-inactivated before it is treated to the transformed cell line for the matter of safety of serum, but not always limited thereto.
  • the present inventors constructed a transformed cell line successfully transfected with a recombinant reporter gene vector comprising a dioxin responsive element (DRE) binding site, a mouse mammary tumor virus (MMTV) derived promoter, and a luciferase gene in the downstream, and analyzed the correlation between dioxin content in human serum and physical variables using the same.
  • DRE dioxin responsive element
  • MMTV mouse mammary tumor virus
  • IGR impaired glucose regulation
  • IGF impaired glucose tolerance
  • IGF impaired fasting glucose
  • DM diabetes mellitus
  • NGT normal glucose tolerance
  • IGR or DM patient demonstrated higher luciferase activity than that of the serum of NGT patient.
  • MetS component metabolic syndrome component
  • fold induction value of dioxin increased
  • fold induction value of dioxin in MetS patient was significantly increased compared with that in normal people. So, it was confirmed that the increase of fold induction value of dioxin raised the risk of diabetes or metabolic syndrome.
  • the significant correlation between dioxin content in serum and diabetes or metabolic syndrome was confirmed. Accordingly, it has been confirmed that the detection method for dioxins of the present invention using the transformed cell line comprising a recombinant reporter gene whose expression is regulated by environmental hormone and total serum can be effectively used for the prediction of the likelihood of diabetes or metabolic syndrome and the prediction of the prognosis.
  • 1.8 kb virus promoter fragment comprising mouse CYP1A1 promoter (482 bp) having 4 DRE binding sites (AhR binding sites) (5′-TNGCGTG-3′) and long terminal repeat (LTR) of mouse mammary tumor virus (MMTV) excluding glucocorticoid response element was cut out from pGudLuc1.1 vector by using HindIII, which was cloned in HindIII site of pGL3-basic vector digested with HindIII. As a result, pCYP1A1-luc vector was constructed (Han et al., BioFactors, 20:11-22, 2004). The direction of the promoter was confirmed by sequencing ( FIG. 1 ).
  • Hepa1c1c7 the mouse hepatocarcinoma cell line, was distributed in a 6 well plate (1 ⁇ 10 5 cells/well), followed by culture for 24 hours until the confluency reached 50%.
  • 2 ⁇ g of pCYP1A1-luc vector constructed in Example ⁇ 1-1> and 0.5 ⁇ g of pcDNA3.1 vector were added to 100 ⁇ l of MEM- ⁇ (serum and antibiotic free) and then mixed with 10 ⁇ l of Superfect (Qiagen), followed by reaction at room temperature for 10 minutes. Then, 600 ⁇ l of MEM- ⁇ supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) was added thereto.
  • FBS fetal bovine serum
  • P/S penicillin/streptomycin
  • the cell line stably expressing pCYP1A1-luc constructed in Example ⁇ 1-2> was distributed in 60 mm culture dish at the density of 1 ⁇ 10 5 cells, followed by culture for 48 hours. Upon completion of the culture, the medium was replaced with DMEM supplemented with 0.5% charcoal-stipped FBS but not supplemented with phenol red, and indole-3-carbinol was treated thereto at the concentrations of 0, 0.01, 0.1, 1, 10, and 100 ⁇ M for 4, 8, or 24 hours. Then, the cells were collected, followed by luciferase assay.
  • luciferase activity of the group treated with 0.001 ⁇ 10 ⁇ M of indol-3-carbinol was increased dose-dependently, compared with that of the non-treated control group.
  • indol-3-carbionol was treated at the concentration of 100 ⁇ M
  • luciferase activity was increased at least 3 times. At this time, significant difference over the time was not detected ( FIG. 3 ).
  • the cell line stably expressing pCYP1A1-luc constructed in Example ⁇ 1-2> was distributed in 60 mm culture dish at the density of 2 ⁇ 10 5 cells, followed by culture for 24 hours. Upon completion of the culture, the medium was replaced with DMEM supplemented with 0.5% charcoal-stipped FBS but not supplemented with phenol red, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was treated thereto at the concentrations of 0, 0.1, 1, 10, 100, and 1000 pM for 4, or 8 hours. Then, the cells were collected, followed by luciferase assay.
  • TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin
  • the group treated with TCDD at the concentration of 0.1 or 1 pM demonstrated similar luciferase activity to that of the control not treated with TCDD, while the group treated with 10 ⁇ 1000 ⁇ M of TCDD demonstrated significantly increased luciferase activity. Luciferase activity was increased over the treatment time, which was not so significant, though ( FIG. 4 ).
  • Serum samples obtained from 97 people were heat-inactivated at 65° C. for 30 minutes. This heat-inactivation process secures the safety of cell culture.
  • the cell line stably expressing pCYP1A1-luc constructed in Example ⁇ 1-2> was distributed in a 96 well plate at the density of 5 ⁇ 10 4 cells/well, followed by culture for 24 hours. The medium was replaced with DMEM not containing phenol red (90 ⁇ l/well), to which 10% human serum sample was treated (10 ⁇ l/well) for 24 hours. Then, the cells of each well were lysed by using luciferase lysis buffer (Promega). The lysate of each well was transferred into a 96 well plate (1 ⁇ l/well), followed by protein quantification by BCA method. The remaining cell lysates proceeded to luciferase assay using Promega luciferase assay kit. The luciferase activity was modified with protein concentration and the results were presented by fold induction or % Control.
  • fold induction values of the serum samples obtained from 97 people were distributed mainly in the range of 2.00 ⁇ 3.00 fold, and the highest rate was detected in approximately 2.50 ⁇ 2.65 fold.
  • the mean fold induction value was approximately 2.35 fold ( FIG. 5 ).
  • Dioxin fold induction value was also investigated in relation to drinking and smoking. As a result, as shown in Table 3 and Table 4, dioxin fold induction value was higher in drinkers/smokers than in non-drinkers/non-smokers.
  • Multivariate analysis was performed by modifying with all the relevant variables. As a result, as shown in Table 5, it was confirmed that only drinking or non-drinking was significantly related thereto.
  • Luciferase activity over dioxin was investigated in relation to normal glucose tolerance (NGT), impaired glucose regulation (IGR) composed of impaired glucose tolerance (IGT) and impaired fasting glucose (IFG), and diabetes mellitus (DM).
  • NGT normal glucose tolerance
  • IGR impaired glucose regulation
  • IGF impaired fasting glucose
  • DM diabetes mellitus
  • Dioxin fold induction was also analyzed over the number of metabolic syndrome component (MetS component). As a result, as shown in Table 7 and Table 8, as the number of MetS component increased, dioxin fold induction value increased. In MetS patients, dioxin fold induction value was significantly increased, compared with that in normal people.
  • the detection system of the present invention characterized by treating total serum to the transformed cell line harboring the recombinant reporter gene whose expression is regulated by dioxin compounds is advantageous in easy and accurate high efficiency detection of dioxins in serum. Therefore, the detection system of the present invention can be effectively applied to diagnostic techniques for the prediction of disease occurrence and the determination of treatability, based on the consideration of the correlation between dioxin compounds and patient's disease factors.

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CN109306001A (zh) * 2018-11-21 2019-02-05 浙江海洋大学 芳香烃受体核转位蛋白arnt——一种新型海洋生物污染检测标志物
CN109813913B (zh) * 2019-01-31 2021-11-09 中国医学科学院肿瘤医院 芳烃受体(AhR)在预测免疫治疗效果中的应用

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