KR100858168B1 - Method of Diagnosis of Toxicity for Polybrominated Diphenyl Ethers using Target organ-array and PBDEs-specific proteins - Google Patents

Abstract

The present invention relates to a diagnostic method using a target organ array and a specific protein for bromine compounds for the exposure and exposure of bromine compounds, more specifically, by selecting organs and proteins that are sensitive to bromine compounds. The present invention relates to a method for diagnosing bromine compound exposure and intoxication by identifying a change in the amount of protein showing a specific change in a target organ.

 The present invention is widely contaminated in the environment by detecting the amount of protein expression of pregrine X receptor or apolipoprotein A-1 of pituitary tissue, Bcl-2 of thyroid tissue, CYP1A1 or UGT1A of hepatic tissue or androgen receptor of prostate tissue, It can be used to diagnose bromine compounds exposure and intoxication that cause endocrine disorders such as decreased intelligence and thyroid dysfunction.

Bromine Compounds, Target Organ Arrays, Specific Proteins

Description

Method of Diagnosis of Toxicity for Polybrominated Diphenyl Ethers using Target organ-array and PBDEs-specific proteins for exposure of bromine compound and diagnosis of intoxication

1 shows a schematic of a set of target organ arrays for diagnosing bromine compounds (PBDEs) exposure and intoxication.

Figure 2a shows the expression of CYP1A1 in the liver when bromine compound poisoning.

Figure 2b shows the expression of UGT1A in liver when bromine compound poisoning.

Figure 3 shows the expression of Bcl-2 in the thyroid gland upon bromine compound poisoning.

Figure 4 shows the AR expression in the prostate when bromine compound poisoning.

Figure 5a shows the expression of PXR in the pituitary gland when bromine compound poisoning.

Figure 5b shows the expression of Apolipoprotein A-1 in the pituitary gland when bromine compound poisoning.

The present invention relates to a diagnostic method using a target organ array and a specific protein for bromine compounds for the exposure and exposure of bromine compounds, more specifically, by selecting organs and proteins that are sensitive to bromine compounds. The present invention relates to a method for diagnosing bromine compound exposure and intoxication by identifying changes in the amount of protein showing specific changes in target organs.

Bromine compounds are hardly decomposable organic halogen compounds produced around 40,000 tons annually worldwide. They are used as flame retardants mixed with various plastic products, wallpaper, floorings, carpets, textiles, and various electrical appliances. With the recent development of the industry, pollution in environmental samples, foods and breast milk continues to increase, which is a problem. In addition to breathing and skin exposure from the environment, humans are exposed mainly by eating foods containing large amounts of fat. In infants and young children, they are exposed through breast milk or milk powder. This is because the bromine compound has high fat affinity and the half-life in the body is long, such as 19-119 days depending on the chemical structure, so that the residence time in the living body is long.

Bromine compounds are suspected carcinogens, endocrine barriers and neurodevelopmental toxins. In particular, bromine compounds cause thyroid dysfunction, and when babies and newborns are exposed through breast milk or milk powder, cognitive decline and behavioral abnormalities are induced. In addition, male maturation is delayed and sperm count and sperm motility have been reported. However, the endocrine toxicity is similar to the toxic symptoms caused by similar environmental pollutants or drugs such as dioxins, polychlorinated biphenyls (PCBs), and propylthiouracil (propylthiouracil). it's difficult.

Bromine compounds have been reported to be more than 100 times higher than in the past 30 years as the pollution of the environment has increased, and that the main source of human exposure is foods containing large amounts of fat such as pork. Exposure to human body causes problems in various organs such as hypothyroidism, delayed maturation, cognitive decline, and nervous system disorders. Toxicity detection and evaluation of each compound is a very important indicator for the diagnosis of intoxication of full compounds. Bromine compounds increase the activity of the uridine diphospho-glucuronosyl transferase (UDPGT), a microsomal phase II enzyme in the liver, and by this enzyme thyroid hormone (T4) Increased metabolism of bile, leading to increased bile thyroidemia, and activating EROD, MROD, and PROD (ethoxy-, methoxy) by activating the enzyme system of CYP1A1 and CYP2B, the microsomal phase I enzymes in the liver. -And pentoxy-resorufin-O-deethylase) increases the metabolic capacity of the liver.

 In order to detect the toxicity of bromine compounds in the body, changes in thyroid hormone and hepatic enzymes are examined, and changes in hormones and metabolic enzymes are not easy to distinguish from toxicity of chemicals such as dioxins and PCBs, which are similar environmental pollutants. It is also difficult to distinguish from the action of drugs such as propylthiouracil. Various endocrine and neurological disorders caused by brominated compounds are suspected of damaging the thyroid gland or brain, but until now the target organs are not clearly identified except the thyroid gland. Other than increased activity of UDPGT, CYP1A1 and CYP2B family enzymes).

Toxicity diagnosis using whole organs when exposure of bromine compounds requires a lot of time, manpower, etc., and pathological histological expertise is important, and it is difficult to carry out easily in a short time. It is composed of more than 200 species of homologous chemicals, so it is not easy to inspect and analyze them individually. In addition, various toxicities such as nervous system, thyroid dysfunction, etc. have been revealed, but the mechanism of toxicity is not clearly revealed, which is a definite diagnostic indicator.

Recently, researches to find biomarkers in vivo have been actively conducted to easily detect changes in vivo in response to external stimuli (exposures caused by diseases, infections, drugs, etc.). Searching for proteins or genes that have been specifically changed by chemicals such as bromine compounds and investigating the extent of their specific changes in each organ will facilitate diagnosis or exposure assessment and can be applied as diagnostic kits. Its utility is very large.

SUMMARY OF THE INVENTION An object of the present invention is to provide a target organ array set for diagnosing bromine compound exposure and intoxication which is sensitive to exposure and toxicity to bromine compounds.

Another object of the present invention is to detect bromine by detecting the expression level of pregrine X receptor or apolipoprotein A-1 of pituitary tissue, Bcl-2 of thyroid tissue, cytochrome P4501A1 or UGT1A of hepatic tissue, and androgen receptor of prostate tissue. To provide a method for diagnosing compound exposure and intoxication.

In order to achieve the above object, the present invention can attach a control sample attached to the surface and a sample to be measured for any one or more selected from the normal group of pituitary tissue, thyroid tissue, liver tissue, and prostate tissue. Provided is a set of target organ arrays for diagnosing bromine compound exposure and intoxication, including attachments under test.

In addition, the present invention provides antipregine X receptor antibody, anti apolipoprotein A-1 antibody, anti Bcl-2 antibody, anti cytochrome P4501A1 antibody, anti UDP-glucuronosyltransferase 1A antibody, anti androgen receptor Provided is a bromine compound exposure and addiction diagnostic set comprising at least one antibody selected from among antibodies.

In addition, the present invention relates to pregrine X receptor or apolipoprotein A-1 of pituitary tissue, Bcl-2 of thyroid tissue, cytochrome P4501A1 or UDP-glucuronosyltransferase 1A of hepatic tissue, and androgens of prostate tissue. It provides a bromine compound exposure and toxicosis diagnostic method characterized by detecting the expression level of any one or more proteins selected from the receptor.

Hereinafter, the present invention will be described in detail.

The present invention is a set of proteins and target organs specifically affected by bromine compounds for the diagnosis and exposure evaluation of bromine compounds that are widely contaminated in the environment and cause endocrine disorders such as intelligence loss and thyroid dysfunction. This was done to examine exposure and intoxication sensitively and accurately.

The present invention is bromine compound, a chemical that is mainly used as a fire retardant in high-flammability products, such as plastic products, as its use increases with the development of the industry, the amount of pollution in living organisms, foods, and the environment increases, so as to increase human exposure, its toxicity and exposure. Since evaluation is important, we tried to develop bromine compound exposure detection and addiction diagnosis method more simply and sensitively using multi-array array technique and protein analysis technique.

The target organ array set for diagnosing bromine compound exposure and intoxication of the present invention comprises a control sample attachment portion and a sample to be measured to which a control group is attached to the surface of at least one selected from the pituitary tissue, thyroid tissue, liver tissue, and prostate tissue of a normal group. It includes an attachment to be measured.

Depending on the organ to be diagnosed for bromine compound exposure and addiction diagnosis, antipregine X receptor antibody, anti apolipoprotein A-1 antibody, anti Bcl-2 antibody, anti cytochrome P4501A1 antibody, anti UDP-glucu One or more antibodies can be selected and used from a lonosyltransferase 1A antibody and an anti-androgen receptor antibody.

Bromine compound exposure and toxicosis diagnostic method of the present invention is a pregnein X receptor or apolipoprotein A-1 of pituitary tissue, Bcl-2 of thyroid tissue, cytochrome P4501A1 or UDP-glucuronosyltransferase of hepatic tissue Bromine compound exposure and intoxication are diagnosed by detecting the amount of protein expression of the androgen receptor of 1A or prostate tissue.

For the exposure of bromine compound and diagnosis of intoxication, the tissues of each organ to be diagnosed are made into paraffin blocks, and the blocks are circumferentially circulated using a pin of about 2 mm diameter and transferred to a new paraffin block. When the tissue and paraffin are completely combined, the microtome is cut into a thickness of about 3 μm and placed on a measuring attachment to which the sample of the target organ array is attached. Put the slide in the citrate buffer of 6 and boil it for 10-15 minutes at 121 ℃ using autoclave. After antigen restoration, the slides were left for 10 minutes in a solution of 3% hydrogen peroxide diluted 1: 5 in methanol to remove peroxidase enzyme.

Antibodies against CYP1A1 or UGT1A in the liver, antibodies against Bcl-2 in the thyroid gland, and antibodies against apolipoprotein A-1 to the pituitary gland were reacted. Afterwards, it is developed using diamino-benzidine (DAB) and the stained nucleus with hematoxylin (Hematoxylin) and fixed to cover the cover slide. Image analysis software (i-Solution) is used to quantify the immunostaining results. CYP1A1, UGT1A, Bcl-2, Pregnane X receptor and Apolipoprotein A-1 are stained in the cytoplasm to give a percentage of the stained area relative to the total area of the androgen receptor. Calculate the thickness of the line tissue as the average of the four measurements.

According to the present invention, bromine compound exposure and intoxication can be determined by increasing the CYP1A1, UGT1A protein of the liver, the pregine X receptor of the pituitary gland, and the apolipoprotein A-1 protein by more than two times, respectively. In addition, when the Bcl-2 of the thyroid and the androgen receptor protein of the prostate are reduced to about 2 times or less compared to the normal group, bromine compound exposure and poisoning can be determined.

As described above, the present invention provides a pregrine X receptor or apolipoprotein A-1 of pituitary tissue, Bcl-2 of thyroid tissue, cytochrome P4501A1 or UDP-glucuronosyltransferase 1A of hepatic tissue, or prostate tissue. Bromine compound exposure and intoxication can be diagnosed by detecting the expression level of any one or more proteins selected from androgen receptors.

Therefore, the bromine compound exposure assessment and the specific protein-target organ set for diagnosing toxicosis not only help to facilitate the diagnosis of toxicosis of bromine compounds and to study the toxicity mechanism, but also be useful for the exposure evaluation.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples. However, these examples are only presented to understand the content of the present invention, but the scope of the present invention is not limited to these embodiments.

Example 1 Administration of Test Animals and Test Drugs

In 28-day-old male rats (Wistar rat: n = 3), DE-71 (mixture of PBDEs, Cambridge Isotope Laboratories Inc.) at 0, 3, 30 and 100 mg / kg body weight was diluted in corn oil. Administration was oral for 2 weeks. After administration, the rats were sacrificed to measure organ weights of the brain, pituitary gland, liver, thyroid, kidney, prostate, testicle, epididymis, adrenal gland, spleen, and fixed with formaldehyde. As a result of long-term gravimetric analysis, the hepatic and thyroid gland weights increased and the prostate weight decreased in the administration group.

Example 2 Target Organ and Specific Protein Selection Method

The tissue fixed in formalin by Example 1 was embedded in a paraffin block after dehydration. Tissue sections were prepared by sectioning each block and hematoxylin and eosin (HE) staining were performed. While looking at the HE staining of the slides of each tissue, using a 2 mm diameter pin, the tissue of each organ (brain, pituitary gland, liver, thyroid, kidney, prostate, testes, epididymis, adrenal gland) embedded in paraffin was circumferentially excavated. The long-term array blocks were prepared by transferring to new paraffin blocks.

Multi-organ array block is 0, 3mg / kg, 30mg / kg, 100mg / by dose of PBDEs for 9 kinds of tissues (brain, pituitary, liver, thyroid, kidney, prostate, testes, epididymis, adrenal gland). A total of four dose groups of ㎏ consisted of a total of 36 different tissues in one block. In order to completely fill the gap between the prepared block paraffin and the newly planted tissue, the block was placed in a thermostat at 58 ° C. and left for one hour. When the tissue and paraffin were completely bonded, the microtomes were used to slice 3 μm thick and placed on the glass slide. After hydration, the slides are placed in a citrate buffer of pH 6 for antigen restoration and boiled at 121 ° C. for 10 minutes using an autoclave. After antigen restoration, the peroxidase enzyme is removed. The slides were left for 10 minutes in a solution of 3% hydrogen peroxide diluted 1: 5 in methanol.

Considering the results of clinical trials such as endocrine disruption induced by bromine compound exposure, the protein is considered to be related to the increase of apolipoprotein A-1 in the blood, the protein that changes during endocrine disruption, carcinogenesis or apoptosis Were divided into three groups of proteins. Group 1 proteins include Apolipoprotein A-1, presenilin 2, PXR, CYP1A1, and UGT1A, and group 2 proteins include AR, PR, ER-α, and EGFR. Six proteins, including c-Myc, PKC-α, PCNA, Bax, Bcl-2, and Cox2, were selected for species and the third group of proteins, and a total of 15 proteins were immunostained. Each selected protein antibody was diluted 1: 100 in PBS dissolved in 5% skim milk, 0.05% Tween-20, and then reacted with slides of 100 μl for 30 minutes, followed by PBS dissolved in 0.05% Tween-20. Antibodies that did not bind by washing were removed. Unbound antibody was washed with PBS dissolved in 0.05% Tween-20 for 20 minutes of the enzyme-bound secondary antibody on a slide of 100 μl using a SuperPicTure ^TM Polymer Detection Kit (Zymed Laboratory Inc., USA). Were removed. After developing with diamino-benzidine (DAB), washed three times with PBS dissolved 0.05% Tween-20, and 0.05% Tween-20 after counter staining with hematoxylin. Washed three times with dissolved PBS. Once the hematozain was cleared, a drop of mounting solution was dropped and the cover slide covered after dehydration. Immunostaining results were quantified using image analysis software (i-Solution).

Immunostaining results for 15 proteins were analyzed to select target organ arrays and specific protein sets by selecting proteins and organs that increased or decreased correlated with the amount of bromine compound exposed (Table 1). As shown in Table 1, the expression levels of CYP1A1 and UGT1A in the hepatic tissue, pregnane X receptor and Apolipoprotein A-1 in the pituitary gland increased with increasing bromine compound concentration. The expression levels of Bcl-2 in thyroid tissue and androgen receptor in prostate tissue tended to decrease with increasing bromine concentration.

<Table 1> Immunostaining results and expression levels of specific proteins quantified by target organs (%)

group protein Normal Administration group (PBDEs 3mg / kg) Administration group (PBDEs 30mg / kg) Administration group (PBDEs 100mg / kg) Soy sauce CYP1A1 0.8 ± 1.4 1.1 ± 1.9 16.6 ± 11.2 26.4 ± 11.6 Soy sauce UGT1A 12.3 ± 3.5 10.9 ± 3.9 24.4 ± 3.6 32.4 ± 14.6 thyroid Bcl-2 1.3 ± 1.5 0.3 ± 0.2 0.8 ± 0.9 0.8 ± 0.6 prostate AR 15.4 ± 8.0 12.5 ± 4.8 7.7 ± 1.9 5.7 ± 3.4 pituitary PXR 2.5 ± 0.7 3.0 ± 1.7 15.8 ± 1.7 16.8 ± 0.3 pituitary Apolipoprotein A-1 6.6 ± 4.7 28.1 ± 14.6 29.3 ± 13.6 30.2 ± 8.9

CYP1A1: cytochrome P4501A1

UGT1A: UDP-glucuronosyltransferase 1A

AR: Androgen receptor

PXR: Pregnane X receptor

Example 3 Constructing, Producing and Specific Reaction Method of Target Organ Array Set

Using a 2 mm diameter pin, tissues of the target organs (pituitary, thyroid, hepatic and prostate) embedded in paraffin were circumferentially excised and transferred to a new paraffin block to prepare a multi-organ array block. In order to completely fill the gap between the newly prepared block paraffin and the newly planted tissue, the block was placed twice in an incubator at 58 ° C. for one hour. When the tissue and paraffin are completely combined, the microtome is cut into 3 μm thickness, placed on a glass slide, and hydrated. The slide is placed in a citrate buffer of pH 6 to restore the antigen. Then boiled at 121 ° C. for 10 minutes.

After antigen restoration, the slides were left for 10 minutes in a solution of 3% hydrogen peroxide diluted 1: 5 in methanol to remove peroxidase enzyme. In order to treat the antibodies to specific proteins, a border was drawn around the tissue using a pop pen as shown in FIG. 1. 5% skim milk with antibodies against CYP1A1 or UGT1A in the liver, antibodies against Bcl-2 in the thyroid gland, and antibodies against PXR and apolipoprotein A-1 in the pituitary gland , 0.05% Tween-20 was diluted 1: 100 in PBS dissolved in 40μL drop on the organ.

Observing the tissues do not dry out, and left for 30 minutes, washed three times with PBS dissolved in 0.05% Tween-20 to remove the unbound antibodies. Using the SuperPicTure ^TM Polymer Detection Kit (Zymed Laboratory Inc., USA), 100 μl of the enzyme-bound secondary antibody was dropped into the tissue and left for 20 minutes, followed by washing three times with PBS in 0.05% Tween-20. Antibodies were removed. After developing with diamino-benzidine (DAB), washed three times with PBS dissolved 0.05% Tween-20, and 0.05% Tween-20 after counter staining with hematoxylin. Wash three times with dissolved PBS. Once the hematozain was cleared, a drop of mounting solution was dropped and the cover slide covered after dehydration.

Immunostaining results were quantified using image analysis software (i-Solution). CYP1A1, UGT1A, Bcl-2, Pregine X Receptor, and Apolipoprotein A-1 were stained in the cytoplasm and the percentage of the stained area relative to the total area, and the Androgen Receptor measured the thickness of the gland tissue in all four areas. Calculated on average.

Example 4 Results Determination and Diagnosis Method

Target organ arrays of bromine compound-administered groups (PBDEs 30 mg / kg body weight or more) were prepared and immunostained with bromine compound-specific proteins, and the expression level of specific proteins was measured based on the untreated control (n = 3). More than two-fold increase in CYP1A1 and UGT1A acting on drug metabolism in the liver, more than two-fold increase in PXR in the pituitary gland and about two-fold increase in apolipoprotein A-1, while in the androgen receptor and thyroid in the prostate When the decrease of Bcl-2 was observed, it was diagnosed that the bromine compound was exposed to more than the amount of toxic expression or toxicosis was expressed. At this time, the image analysis was performed by converting each immunostaining result into a percentage of the area of the immunostained portion of the total area using an image analysis program.

The percentage (immune staining / total area) obtained using the image analysis program was statistically processed and the result judgment and diagnosis were set according to the criteria (Table 2) based on the results for the mean (n = 3) and the error range. .

<Table 2> Criteria for Determination of Immunostaining Results in Bromine Compound Addiction

long time protein Criteria Soy sauce CYP1A1 More than double the normal group Soy sauce UGT1A More than double the normal group thyroid Bcl-2 Less than 1/2 of normal group prostate Androgen receptor Less than 1/2 of normal group pituitary Pregnane X receptor More than double the normal group pituitary Apolipoprotein A-1 More than double the normal group

      ※ To check whether the bromine compound is contaminated more than the toxicity of bromine compounds, immature rats of 4 to 6 weeks of age are used, and the suspected sample is forcibly administered to the rats and tested for expression of the protein.

As a result, when bromine compound poisoning, the expression of CYP1A1 and the expression of UGT1A in the liver increased as shown in FIGS. 2A and 2B, and the expression of Bcl-2 in the thyroid gland decreased as shown in FIG. 3. In addition, androgen receptor expression was decreased in the prostate as shown in FIG. 4, and PXR and apolipoprotein A-1 expression in the pituitary gland was increased as shown in FIGS. 5A and 5B.

As described above, bromine compounds by providing a diagnostic method using organs and specific proteins that specifically react with bromine compounds for the diagnosis of toxicity and poisoning of bromine compounds using multi-organ array and protein analysis technology according to the present invention. Changes in organs and specific proteins that are sensitive to the virus can be performed quickly. In addition, it is easy to confirm the poisoning and contamination of bromine compounds in humans and livestock, which facilitates human health management and assessment of bromine compound exposure in livestock.

Claims (3)

delete Bromine including antipregine X receptor antibody, anti apolipoprotein A-1 antibody, anti Bcl-2 antibody, anti cytochrome P4501A1 antibody, anti UDP-glucuronosyltransferase 1A antibody and anti androgen receptor antibody Set of antibodies for diagnosing compound exposure and intoxication. In the diagnosis of bromine compound exposure and poisoning in livestock, Attaching a normal group of pituitary tissue, thyroid tissue, liver tissue and prostate tissue to the surface as a control sample, and attaching a sample to be measured for the control to create a target organ array set; Reacting the antibody set of claim 2 with the target organ array set; And Pregnane X receptor and apolipoprotein A-1 in pituitary tissue, Bcl-2 in thyroid tissue, cytochrome P4501A1 and UDP-glucuronosyl transferase 1A in hepatic tissue and androgen in prostate tissue Bromine compound exposure and intoxication diagnostic method for livestock, characterized in that for detecting the amount of protein expression of the receptor.

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