U.S. patent application number 15/051385 was filed with the patent office on 2017-04-20 for novel biological detection method for dioxins in serum, and a diagnostic use therefor in metabolic syndrome and related conditions.
The applicant listed for this patent is UNIVERSITY-INDUSTRY COOPERATION GROUP OF KYUNG HEE. Invention is credited to Youngmi Kim, Hong Kyu Lee.
Application Number | 20170107584 15/051385 |
Document ID | / |
Family ID | 45811063 |
Filed Date | 2017-04-20 |
United States Patent
Application |
20170107584 |
Kind Code |
A1 |
Lee; Hong Kyu ; et
al. |
April 20, 2017 |
NOVEL BIOLOGICAL DETECTION METHOD FOR DIOXINS IN SERUM, AND A
DIAGNOSTIC USE THEREFOR IN METABOLIC SYNDROME AND RELATED
CONDITIONS
Abstract
The present invention relates to a novel biological detection
method for dioxins in serum and a diagnostic use thereof in
metabolic syndrome and related conditions. More particularly,
according to the present invention, a significant correlation
between dioxin content in serum and physical variables has been
confirmed by measuring luciferase activity through the use of serum
as a whole and cells transformed with a recombinant vector
comprising a gene construct in which a dioxin responsive element, a
promoter and a reporter gene are operably linked. And there is an
improvement in this method of the present invention over the
conventional methods which require a preprocessing step in order to
purify dioxins from serum, by which plural samples can be analyzed
easily and accurately even with a very small amount of each because
of the use of serum as a whole. Therefore, the detection system of
the present invention which involves total serum processing in a
genetically transformed cell line comprising a recombinant reporter
gene whose expression is modified by dioxin compounds can be
effectively used for the biological detection of dioxins in serum.
This method can also be used to research the correlation between
specific POPs and patient's disease factors by accurate detection
of POPs such as dioxins in the serum, and further this method can
be effectively used to predict the occurrence of disease and to
determine treatability.
Inventors: |
Lee; Hong Kyu; (Yongsan-gu,
KR) ; Kim; Youngmi; (Gangnam-gu, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY-INDUSTRY COOPERATION GROUP OF KYUNG HEE |
Gyeonggi-do |
|
KR |
|
|
Family ID: |
45811063 |
Appl. No.: |
15/051385 |
Filed: |
February 23, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13821779 |
Mar 8, 2013 |
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PCT/KR2011/006583 |
Sep 6, 2011 |
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15051385 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5308 20130101;
C12Q 1/66 20130101; G01N 2800/04 20130101; C12Q 1/6897
20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 10, 2010 |
KR |
10-2010-0088905 |
Claims
1. A detection method for a dioxin compound in serum comprising the
following steps: i) preparing a transformed cell line by
introducing a recombinant vector comprising a gene construct, in
which at least one of dioxin-responsive elements (DRE) represented
by SEQ. ID. NO: 1, a promoter, and a reporter gene are operably
linked, into a host cell; ii) culturing the transformed cell line
prepared in step i) after treating the cell line with the serum of
a test subject; iii) detecting the expression of the protein
expressed by the reporter gene in the transformed cell line
cultured in step ii); and iv) determining the serum to have a
dioxin compound when the reporter gene expression is detected in
step iii).
2. A method of increasing the detection efficiency for a dioxin
compound in serum comprising the following steps: i) obtaining a
serum sample by heat-inactivating total serum obtained from a test
subject; ii) preparing a transformed cell line by introducing a
recombinant vector comprising a gene construct, in which at least
one dioxin-responsive element (DRE) represented by SEQ. ID. NO: 1
is operably linked to a promoter and a reporter gene, into a host
cell; iii) culturing the transformed cell line prepared in step ii)
with the serum sample obtained in step i); iv) detecting the
activation of the reporter gene in the transformed cell line
cultured in step iii); and v) determining that the dioxin compound
is comprised in the serum sample when the activation of the
reporter gene is detected in step iv).
3. The method according to claim 2, wherein the detection
efficiency is determined based on a small amount of serum.
4. The method of claim 3, wherein the small amount of serum is 1-10
.mu.l.
5. The method of claim 2, wherein the dioxin compound is selected
from the group consisting of polychlorinated dibenzodioxins
(PCDDs), polychlorinated dibenzo-furans (PCDFs), polychlorinated
biphenyls (PCBs), polycyclic aromatic hydrocarcons (PAHs),
flavonoids, and pesticides.
6. The method of claim 2, wherein between about 3-4 of the
dioxin-responsive elements of step i) are included in the gene
construct.
7. The method of claim 2, wherein the promoter of step ii) is
selected from the group consisting of MMTV (Mouse Mammary Tumor
Virus) promoter, SV40 promoter, and CMV (cytomegalovirus)
promoter.
8. The method of claim 2, wherein the reporter gene of step ii) is
selected from the group consisting of luciferase, alkaline
phosphatase, chloramphenicol acetyl transferase, and
.beta.-galactosidase.
9. The method of claim 2, wherein the host cell of step ii) is a
mammalian tumor cell line.
10. The method of claim 9, wherein the host cell is a mouse
hepatocarcinoma cell line.
11. The method of claim 2, wherein diabetes or metabolic syndrome
is predicted when the serum sample is the serum of a test subject
suspected to have diabetes or metabolic syndrome.
12. The method of claim 2, wherein a prognosis of diabetes or
metabolic syndrome is monitored when the serum sample is the serum
of a test subject suspected to have diabetes or metabolic syndrome.
Description
RELATED APPLICATIONS
[0001] This application is to a divisional of U.S. patent
application Ser. No. 13/821,779, filed on Mar. 8, 2013, which is a
national stage application under 35 U.S.C. .sctn.371 of
International Patent Application No. PCT/KR2011/006583, filed on
Sep. 6, 2011, which claims priority to Korean Patent Application
No. 10-2010-0088905, filed on Sep. 10, 2010. Each of these
applications is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] 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.
[0004] 2. Description of the Related Art
[0005] 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.
Accordingly, many reports have been made saying that blood levels
of various POPs such as dioxins, polychlorinated biphenyls, and
organic phosphorous pesticides are closely related to the
development of insulin resistance, diabetes, obesity, dyslipidemia,
or hypertension (Lee D H et al., Diabetes Care, 2006,
29(7):1638-44; Lee D H et al., Diabetes Care, 2007, March;
30(3):622-8; Lee D H et al., Diabetologia, 2007 September;
50(9):1841-51) among many other illensses.
[0006] Dioxin compounds, one of the most representative POPs, are
called environmental hormone or endocrine disrupter, which have
been important issues world widely. Particularly,
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic
compound among many environmental hormones and among many
artificially synthesized compounds, which is chemically very stable
and lasts almost forever in the nature. Once the compound is inside
of human body, it is accumulated in fat tissues due to its fat
solubility.
[0007] Some of the compound taken in human body is excreted through
urine and bile juice, but the compound is not easily decomposed or
excreted in general. In the mouse treated with TCDD, ubiquinone
(CoQ) in the liver was reduced and ATP synthesis was suppressed
(Toxicol. Appl. Pharmacol. 217: 363, 2006). TCDD induced
AhR-dependent ROS generation, resulting in the decrease of
mitochondria membrane potential (.DELTA..psi.m) with inhibiting the
functions of mitochondria, and hence metabolic syndrome including
diabetes was expected to be developed.
[0008] Once 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. As a
result, 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). When 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.
[0009] It is possible to classify the patients having disease
induced by POPs and to analyze the correlation of elimination of
such compounds and treatment effect by measuring the concentration
of TCDD equivalent (TCDDEq) among many POPs existing in human
serum. As a method for measuring dioxin, 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. However,
for high-throughput assay, high price has to be paid. In addition,
a large amount of serum sample (25.about.200 ml) is required for
the analysis as well. In the meantime, 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. Once the cells having DRE and
luciferase gene are exposed on dioxin, dioxin is bound to
intracellular AhR and ARNT, which is then bound to DRE in the
nucleus. Then, luciferase expression is induced thereby and
accordingly luciferase is synthesized in proportion to the content
of exposed dioxin. Therefore, the changes of luciferase activity
can be effectively used as a detection index for dioxin. By the
said method, dioxin-like compounds can be measured and TCDD
equivalency (TEQ) can also be measured. However, with the recent
technology, 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 me of serum is
necessary, and generally 10.about.20 ml of sample is required).
When 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. In addition, 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. Then, dioxin is extracted. Because of the above
dioxin extraction and purification process, there is a big
difference between the assay value of TCDD standard material and
the quantification value of serum TEQ (Michael, H. et al, 2000,
Toxicol. Sciences, 54: 183-193).
[0010] 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.
[0011] Thus, 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.
As a result, unlike the conventional method requiring pre-treatment
step for the purification of dioxin from serum, 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.
Recently, 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.
SUMMARY OF THE INVENTION
[0012] It is an object of the present invention to provide a novel
biological detection method for dioxins in serum and a diagnostic
use thereof in metabolic syndrome and related conditions.
[0013] To achieve the above object, the present invention provides
a novel biological detection method for dioxins in serum, which
comprises the following steps:
[0014] 1) preparing a transformed cell line by introducing a
recombinant vector comprising a gene construct, in which at least
one of dioxin-responsive elements (DRE) represented by SEQ. ID. NO:
1, a promoter, and a reporter gene are operably linked, into a host
cell;
[0015] 2) culturing the transformed cell line prepared in step 1)
after treating the cell line with the serum of a test subject;
[0016] 3) detecting the expression of the protein expressed by the
reporter gene in the transformed cell line cultured in step 2);
and
[0017] 4) determining the serum to have dioxin compounds when the
reporter gene expression is detected in step 3).
[0018] The present invention also provides a method for predicting
the likelihood of diabetes or metabolic syndrome, which comprises
the following steps:
[0019] 1) preparing a transformed cell line by introducing a
recombinant vector comprising a gene construct, in which at least
one of dioxin-responsive elements (DRE) represented by SEQ. ID. NO:
1, a promoter, and a reporter gene are operably linked, into a host
cell;
[0020] 2) culturing the transformed cell line prepared in step 1)
after treating the cell line with the serum of a test subject;
[0021] 3) detecting the expression of the protein expressed by the
reporter gene in the transformed cell line cultured in step 2);
and
[0022] 4) determining the likelihood of diabetes or metabolic
syndrome when the reporter gene expression is detected in step
3).
[0023] The present invention also provides a method for monitoring
the prognosis of diabetes or metabolic syndrome, which comprises
the following steps:
[0024] 1) preparing a transformed cell line by introducing a
recombinant vector comprising a gene construct, in which at least
one of dioxin-responsive elements (DRE) represented by SEQ. ID. NO:
1, a promoter, and a reporter gene are operably linked, into a host
cell;
[0025] 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;
[0026] 3) detecting the expression of the protein expressed by the
reporter gene in the transformed cell line cultured in step 2);
and
[0027] 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.
[0028] In addition, the present invention provides a preparation
method of a foreign protein with the original form.
Advantageous Effect
[0029] As explained hereinbefore, 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. In addition, 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The application of the preferred embodiments of the present
invention is best understood with reference to the accompanying
drawings, wherein:
[0031] FIGS. 1A-B are a set of diagrams illustrating the cloning of
pCYP1A1-luc vector. FIG. 1A is a picture of an agarose gel
illustrating the cloning of the pCYP1A1-luc vector. FIG. 1B depicts
a vector map of the pCYP1A1-luc vector.
[0032] FIG. 2 is a photomicrograph showing the colonies of
Hepa1C1c7 cell line expressing pCYP1A1-luc stably.
[0033] FIG. 3 is a graph illustrating the results of
indole-3-carbinol response test with HePa1C1c7 cell line expressing
pCYP1A1-luc stably.
[0034] 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.
[0035] FIG. 5 is a graph illustrating the distribution of dioxin
fold induction values.
[0036] FIG. 6 is a graph illustrating the dioxin fold induction
value in human serum over the body weight analyzed by linear
regression analysis.
[0037] FIG. 7 is a graph illustrating the dioxin fold induction
value in human serum over the bmi analyzed by linear regression
analysis.
[0038] FIG. 8 is a graph illustrating the dioxin fold induction
value in human serum over the we analyzed by linear regression
analysis.
[0039] FIG. 9 is a graph illustrating the dioxin fold induction
value in human serum over the sbp analyzed by linear regression
analysis.
[0040] FIG. 10 is a graph illustrating the dioxin fold induction
value in human serum over the tg analyzed by linear regression
analysis.
[0041] FIG. 11 is a graph illustrating the dioxin fold induction
value in human serum over the fbs analyzed by linear regression
analysis.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0042] Hereinafter, the present invention is described in
detail.
[0043] 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.
[0044] Particularly, the said detection method is composed of the
following steps, but not always limited thereto:
[0045] 1) preparing a transformed cell line by introducing a
recombinant vector comprising a gene construct, in which at least
one of dioxin-responsive elements (DRE) represented by SEQ. ID. NO:
1, a promoter, and a reporter gene are operably linked, into a host
cell;
[0046] 2) culturing the transformed cell line prepared in step 1)
after treating the cell line with the serum of a test subject;
[0047] 3) detecting the expression of the protein expressed by the
reporter gene in the transformed cell line cultured in step 2);
and
[0048] 4) determining the likelihood of diabetes or metabolic
syndrome when the reporter gene expression is detected in step
3).
[0049] 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.
[0050] In this method, at least one of the said dioxin-responsive
elements of step 1) is included and most preferably 3.about.4 DREs
are included, but not always limited thereto.
[0051] In this method, 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.
[0052] 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.
[0053] In this method, the reporter gene of step 1) is preferably
selected from the group consisting of luciferase, alkaline
phosphatase, chloramphenicol acetyl transferase, and
.beta.-galactosidase, and is more preferably luciferase if
considering convenience and sensitivity of activity analysis, but
not always limited thereto.
[0054] 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.
[0055] 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.
[0056] When dioxin is introduced into the cell, it is specifically
bound to arylhydrocarbon receptor (AhR), which is introduced into
the nucleus. The dioxin-receptor complex introduced in the nucleus
is then conjugated to ARNT (AhR nuclear translocator), which is
turned into DNA bindable form. Therefore, any cell line that can
express ARNT and AhR endogenously can be used as a host cell
line.
[0057] The transformation herein can be performed by
electroporation, plasmogamy, calcium phosphate (CaPO.sub.4)
precipitation, calcium chloride (CaCl.sub.2) precipitation,
agitation using silicon carbide fiber, or lipofectamine mediated
method, but not always limited thereto.
[0058] 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. However, in the method of the present invention, the serum
of step 2) is total serum, indicating the serum as a whole can be
used.
[0059] 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.
[0060] In a preferred embodiment of the present invention, the
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 the mouse mammary tumor
virus (MMTV) long terminal repeat (LTP) (see FIGS. 1A and 1B) were
constructed. Then, Hepa1c1c7, the mouse hepatocarcinoma cell line,
was transfected with the same to construct the cell line that can
stably express the said recombinant reporter gene (see FIG. 2).
With the constructed cell line, 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) response test was performed. As a result, compared with the
control not treated with TCDD, luciferase activity was
significantly increased when TCDD was treated thereto at the
concentration of 10.about.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.
[0061] 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. To
prepare serum samples, serums were obtained from 97 people,
followed by heat inactivation at 65.degree. 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.
[0062] 10 .mu.l of the serum sample prepared above was treated to
the cell line stably expressing the recombinant reporter gene,
followed by luciferase assay to analyze the correlation between
luciferase activity and physical variables. As a result, fold
induction values of the serum samples obtained from 97 people were
distributed mainly in the range of 2.00.about.3.00, and the highest
rate was detected in approximately 2.50.about.2.65 fold. The mean
fold induction value was approximately 2.35 fold (see FIG. 5).
[0063] It was also confirmed by linear regression analysis that
there was a significant correlation between dioxin content and many
variables factors such as human weight, body mass index (bmi),
waist circumference (wc), systolic blood pressure (sbp), diastolic
blood pressure (dbp), triglyceride (TG), and fasting blood sugar
(fbs). As the values of such factors increased, dioxin fold
induction value also increased (see FIG. 6.about.FIG. 11). Dioxin
fold induction value was higher in drinkers/smokers than in
non-drinkers/non-smokers. After modifying with all the relevant
variables through multivariate analysis, it was confirmed that only
drinking or non-drinking was significantly related thereto. It was
also confirmed by comparing luciferase levels in serums among
impaired glucose regulation (IGR) composed of impaired glucose
tolerance (IGT) and impaired fasting glucose (IFG), and diabetes
mellitus (DM) and normal glucose tolerance (GNT) that luciferase
activity was higher in IGR or DM serum than in NGT serum. In the
meantime, there was no significant difference between IGR serum and
DM serum. Dioxin fold induction over the number of metabolic
syndrome component (MetS component) was also analyzed. As a result,
as the number of MetS component increased, dioxin fold induction
value increased, and the dioxin fold induction value in MetS
patient was significantly increased compared with that in normal
people. It was additionally analyzed whether or not the increase of
dioxin fold induction was related to the risk of metabolic syndrome
or diabetes. As a result, as fold induction value of dioxin
increased by 1, the risk of metabolic syndrome and diabetes
increased 19.7 times and 11.9 times respectively. Even after the
value was modified with bmi, the risk of metabolic syndrome and
diabetes was still increased 13 times and 8.7 times respectively as
the fold induction value of dioxin was increased by 1. After the
value was modified with age, gender, and bmi together, the risk of
metabolic syndrome or diabetes was still increased as the fold
induction value of dioxin was increased.
[0064] Based on the above results, when 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. On
the other hand, the present invention is advantageous in saving
time, costs, and labor by analyzing serum simply heat-inactivated.
In the matter of amount of serum necessary for the analysis, 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.
TABLE-US-00001 TABLE 1 High-sensitive CALUX analysis CALUX analysis
CALUX analysis system (1) system (2) system Reference Garrison P M
et Han D et al. Present Invention al (1996) Fund. (2004) Appl.
Toxicol. BioFactors 30: 194-203 20: 11-22 Transformation Transient
Stable Stable Vector pGudLuc1.1 pGL2-basic pGL3-basic Pretreatment
of Dissolved in Dissolved in Heat-inactivation serum DMSO after
DMSO after hexane extraction hexane extraction Serum amount 10~20
ml 1~10 ml 10 .mu.l for analysis Screening 24-well system 96-well
system 96-well system system Cell line Hepa1c1c7 H1L1.6 mouse
Hepa1c1c7 mouse hepatocarcinoma mouse hepatocarcinoma cell line
hepatocarcinoma cell line cell line Sensitivity in 1 pM~1 nM 1 pM~1
nM 1 pM~1 nM human blood
[0065] Therefore it was confirmed that the dioxin content in serum
detected by the detection method for dioxins of the present
invention was significantly correlated with various physical
variables. Unlike the existing methods requiring a preprocessing
step for purifying dioxins from a sample, 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. In addition, 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.
[0066] 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.
[0067] Particularly, the method for predicting is preferably
composed of the following steps, but not always limited
thereto:
[0068] 1) preparing a transformed cell line by introducing a
recombinant vector comprising a gene construct, in which at least
one of dioxin-responsive elements (DRE) represented by SEQ. ID. NO:
1, a promoter, and a reporter gene are operably linked, into a host
cell;
[0069] 2) culturing the transformed cell line prepared in step 1)
after treating the cell line with the serum of a test subject;
[0070] 3) detecting the expression of the protein expressed by the
reporter gene in the transformed cell line cultured in step 2);
and
[0071] 4) determining the likelihood of diabetes or metabolic
syndrome when the reporter gene expression is detected in step
3).
[0072] 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.
[0073] Particularly, the method for monitoring is preferably
composed of the following steps, but not always limited
thereto:
[0074] 1) preparing a transformed cell line by introducing a
recombinant vector comprising a gene construct, in which at least
one of dioxin-responsive elements (DRE) represented by SEQ. ID. NO:
1, a promoter, and a reporter gene are operably linked, into a host
cell;
[0075] 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;
[0076] 3) detecting the expression of the protein expressed by the
reporter gene in the transformed cell line cultured in step 2);
and
[0077] 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.
[0078] In this method, at least one of the said dioxin-responsive
elements of step 1) is included and most preferably 3.about.4 DREs
are included, but not always limited thereto.
[0079] In this method, 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.
[0080] In this method, the reporter gene of step 1) is preferably
selected from the group consisting of luciferase, alkaline
phosphatase, chloramphenicol acetyl transferase, and
.beta.-galactosidase, and is more preferably luciferase if
considering convenience and sensitivity of activity analysis, but
not always limited thereto.
[0081] 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.
[0082] The transformation herein can be performed by
electroporation, plasmogamy, calcium phosphate (CaPO.sub.4)
precipitation, calcium chloride (CaCl.sub.2) precipitation,
agitation using silicon carbide fiber, or lipofectamine mediated
method, but not always limited thereto.
[0083] In this method, 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.
[0084] 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.
[0085] 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. As a
result, the significant correlation between dioxin content and
human weight, bmi, wc, sbp, dbp, tg, or fbs was confirmed, and
further the significant correlation between dioxin content and
impaired glucose regulation (IGR) consisting impaired glucose
tolerance (IGT) and impaired fasting glucose (IFG), diabetes
mellitus (DM), and normal glucose tolerance (NGT) was also
confirmed. In particular, the serum of IGR or DM patient
demonstrated higher luciferase activity than that of the serum of
NGT patient. As the number of metabolic syndrome component (MetS
component) increased, fold induction value of dioxin increased, and
the 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.
[0086] Therefore, 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.
[0087] Practical and presently preferred embodiments of the present
invention are illustrative as shown in the following Examples,
Experimental Examples and Manufacturing Examples.
[0088] However, it will be appreciated that those skilled in the
art, on consideration of this disclosure, may make modifications
and improvements within the spirit and scope of the present
invention.
Example 1: Construction of a Cell Line Expressing a Recombinant
Reporter Gene Stably
<1-1> Construction of a Recombinant Reporter Gene Vector
[0089] 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 Hind III, which was cloned
in Hind III site of pGL3-basic vector digested with Hind III. 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. 1B).
<1-2> Construction of a Transformed Cell Line Expressing the
Recombinant Reporter Gene
[0090] Hepa1c1c7, the mouse hepatocarcinoma cell line, was
distributed in a 6 well plate (1.times.10.sup.5 cells/well),
followed by culture for 24 hours until the confluency reached 50%.
2 .mu.g of pCYP1A1-luc vector constructed in Example <1-1>
and 0.5 .mu.g of pcDNA3.1 vector were added to 100 .mu.l of
MEM-.alpha. (serum and antibiotic free) and then mixed with 10
.mu.l of Superfect (Qiagen), followed by reaction at room
temperature for 10 minutes. Then, 600 .mu.l of MEM-.alpha.
supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin/streptomycin (P/S) was added thereto. 700 .mu.l of
Superfect-DNA reaction solution was added to Hepa1c1c7 cells washed
with DPBS, followed by culture at 37.degree. C. for 3 hours. Upon
completion of the culture, the cells were washed with DPBS and the
medium was replaced with 2 me of fresh MEM-a supplemented with 10%
FBS and 1% P/S. 300 .mu.l of G418 was added thereto from 24 hours
after the reaction began. G418 resistant cells were collected for 3
weeks. Once colony was formed, each colony was transferred onto 60
mm culture plate (FIG. 2).
Example 2: Response Test with the Transformed Cell Line Expressing
the Recombinant Reporter Gene
<2-1> Response Test Using Indole-3-Carbinol
[0091] The cell line stably expressing pCYP1A1-luc constructed in
Example <1-2> was distributed in 60 mm culture dish at the
density of 1.times.10.sup.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 .mu.M
for 4, 8, or 24 hours. Then, the cells were collected, followed by
luciferase assay.
[0092] As a result, as shown in FIG. 3, luciferase activity of the
group treated with 0.001.about.10 .mu.M of indol-3-carbinol was
increased dose-dependently, compared with that of the non-treated
control group. In particular, when indol-3-carbionol was treated at
the concentration of 100 .mu.M, luciferase activity was increased
at least 3 times. At this time, significant difference over the
time was not detected (FIG. 3).
<2-2> Response test using
2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD)
[0093] The cell line stably expressing pCYP1A1-luc constructed in
Example <1-2> was distributed in 60 mm culture dish at the
density of 2.times.10.sup.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.
[0094] As a result, as shown in FIG. 4, 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.about.1000 pM of TCDD demonstrated
significantly increased luciferase activity.
[0095] Luciferase activity was increased over the treatment time,
which was not so significant, though (FIG. 4).
Example 3: Detection of Dioxins in Human Serum
<3-1> Preparation of Human Serum Samples
[0096] Serum samples obtained from 97 people were heat-inactivated
at 65.degree. C. for 30 minutes. This heat-inactivation process
secures the safety of cell culture.
<3-2> Detection of Dioxins in Human Serum and Analysis of the
Correlation Between Dioxins and Physical Variables
[0097] The cell line stably expressing pCYP1A1-luc constructed in
Example <1-2> was distributed in a 96 well plate at the
density of 5.times.10.sup.4 cells/well, followed by culture for 24
hours. The medium was replaced with DMEM not containing phenol red
(90 .mu.l/well), to which 10% human serum sample was treated (10
.mu.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 .mu.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.
[0098] As a result, as shown in FIG. 5, fold induction values of
the serum samples obtained from 97 people were distributed mainly
in the range of 2.00.about.3.00 fold, and the highest rate was
detected in approximately 2.50.about.2.65 fold. The mean fold
induction value was approximately 2.35 fold (FIG. 5). The
correlation between dioxins and various physical variables was
analyzed. As a result, r and P values of each variable are as shown
in Table 2. Particularly, as shown in FIG. 6.about.FIG. 11, it was
also confirmed by linear regression analysis that there was a
significant correlation between dioxin content and many variables
factors such as human weight, body mass index (bmi), waist
circumference (wc), systolic blood pressure (sbp), diastolic blood
pressure (dbp), triglyceride (TG), and fasting blood sugar (fbs).
As the values of such variables increased, fold induction values of
dioxin also increased (FIG. 6.about.FIG. 11).
TABLE-US-00002 TABLE 2 r P value Age 0.0747397 0.466872841 Weight
0.3287115 0.001011469 Body mass index (bmi) 0.3385887 0.000740128
Waist circumference (wc) 0.4194761 2.0988E-05 Systolic blood
pressure (sbp) 0.3163346 0.001596079 Diastolic blood pressure (dbp)
0.2696278 0.007568065 Serum creatinine (cr) 0.1057983 0.302361753
White blood cell (wbc) 0.185271 0.06924526 got -0.058096
0.571911012 gpt 0.0216235 0.833487555 Cholesterol (chol) 0.1860382
0.068080471 Triglyceride (tg) 0.2508027 0.013218103 High density
lipoprotein (hdl) -0.1656577 0.104886065 Low density lipoprotein
(ldl) 0.1170971 0.253347357 Fasting blood sugar (fbs) 0.2320468
0.022191056 Hemoglobin A1c (hba1c) 0.1845205 0.070400124
[0099] 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.
TABLE-US-00003 TABLE 3 Std. Std. Error Drinking N Mean Deviation
Mean Fold No 43 2.1425 0.49264 0.07513 Yes 50 2.5111 0.54727
0.07740 * P = 0.001
TABLE-US-00004 TABLE 4 Std. Std. Error Smoking N Mean Deviation
Mean Fold No 64 2.2448 0.55652 0.06956 Yes 29 2.5652 0.49577
0.09206 * P = 0.009
[0100] 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.
TABLE-US-00005 TABLE 5 Unstandardized Coefficients Standardized
Sig. Std. Coefficients t Std. Model B Error Beta B Error 1 Constant
0.396 0.534 0.742 0.460 bmi 0.031 0.017 0.188 1.878 0.064 sbp 0.006
0.004 0.171 1.666 0.099 Smoking/ 0.090 0.125 0.076 0.719 0.474
Non-smoking Drinking/ 0.290 0.108 0.263 2.682 0.009 Non-drinking tg
0.000 0.001 0.086 0.834 0.407 fbs 0.002 0.003 0.068 0.660 0.511
[0101] 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). As a result, as
shown in Table 6, luciferase activity was higher in the serum of
IGR or DM than in the serum of NGT. However, there was no
significant difference between the serums of IGR and DM.
TABLE-US-00006 TABLE 6 IGR DM DM vs. vs. vs. NGT IGR DM NGT NGT IGT
N = 50 N = 24 N = 23 p p p Mean SD Mean SD Mean SD value value
value Age 44.1 10.3 49.0 9.3 49.3 8.5 0.052 0.037 0.894 bmi 23.2
3.5 24.7 3.1 25.6 2.4 0.084 0.004 0.246 wc 80.8 7.8 85.7 9.2 87.3
7.6 0.022 0.002 0.508 sbp 116.5 11.4 130.1 16.4 129.7 16.9 0.000
0.000 0.930 dbp 68.5 8.3 76.0 9.7 77.3 11.6 0.001 0.000 0.677 bun
14.4 3.7 14.1 3.5 14.5 3.5 0.705 0.868 0.635 cr 0.8 0.2 0.8 0.2 0.9
0.1 0.771 0.111 0.140 hb 14.5 1.5 14.3 1.7 14.6 1.6 0.500 0.771
0.426 wbc 6.3 1.6 6.5 1.5 6.1 1.3 0.551 0.669 0.341 plt 22.7 4.8
24.0 5.5 24.0 4.9 0.289 0.290 0.978 got 23.4 6.1 23.6 7.4 25.1 7.2
0.880 0.296 0.495 gpt 23.5 8.2 23.3 13.8 27.7 17.5 0.935 0.159
0.333 ggt 27.5 17.1 40.5 39.8 56.3 43.6 0.052 0.005 0.202 chol
198.1 31.9 193.0 34.7 217.6 38.0 0.531 0.025 0.025 tg 112.2 95.2
144.9 118.5 171.9 80.1 0.206 0.011 0.367 hdl 59.8 14.2 56.0 14.9
53.6 12.7 0.299 0.081 0.558 ldl 115.9 30.3 108.0 35.1 129.6 32.4
0.322 0.083 0.034 fbs 90.0 5.6 104.5 4.3 114.5 37.1 0.000 0.005
0.211 hba1c 6.1 0.2 6.1 0.3 7.1 1.2 0.692 0.001 0.001 Fold 2.05
0.47 2.58 0.42 2.77 0.46 0.000 0.000 0.152 (calux)
[0102] 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.
TABLE-US-00007 TABLE 7 MetS component Std. number Mean N Deviation
0.00 2.0191 25 0.47701 1.00 2.1790 23 0.44884 2.00 2.4048 24
0.43824 3.00 2.8263 16 0.47832 4.00 2.7213 6 0.71194 5.00 2.6845 2
0.17577 Total 2.3461 96 0.54742 * P < 0.001, linear regression
analysis
TABLE-US-00008 TABLE 8 Std. Std. Error MetS N Mean Deviation Mean
.00 72 2.1987 0.47686 0.05620 1.00 24 2.7882 0.51367 0.10485 * P
< 0.001, t test
[0103] In addition, it was also investigated how the increase of
dioxin fold induction was related to the risk of diabetes or
metabolic syndrome. As a result, as shown in Table 9.about.Table
11, every time dioxin fold induction value was increased by 1, the
risks of metabolic syndrome and diabetes were increased 19.7 times
and 11.9 times respectively. After modifying the result with bmi,
every time dioxin fold induction value was increased by 1, the
risks of metabolic syndrome and diabetes were still significantly
increased 13 times and 8.7 times respectively. After modifying the
result with age, gender, and bmi together, the risks of metabolic
syndrome and diabetes were still increased.
TABLE-US-00009 TABLE 9 B S.E. Wald df Sig. Exp(B) Metabolic Step
fold 2.981 0.776 14.742 1 0.000 19.701 syndrome 1(a) Constant
-8.565 2.033 17.750 1 0.000 0.000 Diabetes Step fold 2.478 0.686
13.066 1 0.000 11.920 1(a) Constant -7.374 1.802 16.742 1 0.000
0.001
TABLE-US-00010 TABLE 10 B S.E. Wald df Sig. Exp(B) Metabolic Step
fold 2.577 0.808 10.160 1 0.001 13.154 syndrome 1(a) bmi 0.260
0.109 5.701 1 0.017 1.298 Constant -14.037 3.266 18.475 1 0.000
0.000 Diabetes Step fold 2.173 0.702 9.574 1 0.002 8.788 1(a) bmi
0.101 0.097 1.077 1 0.299 1.106 Constant -9.137 2.656 11.836 1
0.001 0.000
TABLE-US-00011 TABLE 11 B S.E. Wald df Sig. Exp(B) Metabolic Step
1(a) fold 2.422 0.837 8.375 1 0.004 11.264 syndrome bmi 0.294 0.116
6.426 1 0.011 1.342 Age 0.040 0.031 1.727 1 0.189 1.041 Gender
0.260 0.642 0.163 1 0.686 1.296 Constant -16.789 4.135 16.488 1
0.000 0.000 Diabetes Step 1(a) fold 1.961 0.707 7.701 1 0.006 7.105
bmi 0.103 0.102 1.026 1 0.311 1.109 Age 0.036 0.029 1.536 1 0.215
1.037 Gender -.437 0.591 0.547 1 0.459 0.646 Constant -9.753 3.244
9.041 1 0.003 0.000
INDUSTRIAL APPLICABILITY
[0104] As explained hereinbefore, 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.
[0105] Those skilled in the art will appreciate that the
conceptions and specific embodiments disclosed in the foregoing
description may be readily utilized as a basis for modifying or
designing other embodiments for carrying out the same purposes of
the present invention. Those skilled in the art will also
appreciate that such equivalent embodiments do not depart from the
spirit and scope of the invention as set forth in the appended
Claims.
Sequence CWU 1
1
117DNAMus musculusmisc_feature(2)..(2)n is a, c, g, or t 1tngcgtg
7
* * * * *