WO2007004361A1 - Transfected non-human mammal and method for monitoring toxic chemical substance in environment using the same - Google Patents

Abstract

Disclosed is a transfected non-human mammal which enables to detect an endocrine disruptor, which is an environmentally toxic substance contaminated in the environment, a part of carcinogens or the like readily at low cost and also enables to monitor such a substance continuously. A non-human mammal (e.g., mouse) having a gene (MMTV-DRE-SEAP) produced by ligating a secreted alkaline phosphatase (SEAP) gene downstream to a gene highly responsive to a dioxin (MMTV-DRE) introduced therein so that alkaline phosphatase is secreted in a body fluid of the mammal when the mammal is exposed to an environment having a toxic chemical substance contaminated therein.

Description

 Transgenic non-human mammal and monitoring method for harmful chemical substances in the environment using the same

 Technical field

 [0001] The present invention reacts with endocrine disrupting substances such as dioxins and polycyclic aromatic hydrocarbons and some carcinogenic substances, which are harmful chemical substances in the environment, and produces a secreted alkaline phosphatase (SEAP). The present invention relates to transgenic non-human mammals that produce and secrete) and methods for monitoring harmful chemicals in the environment that are used to detect and quantify or monitor harmful chemical substances in the environment.

 Background art

 Dioxins, polycyclic aromatic hydrocarbons, and the like have various effects on human health.

 It is known that the action of these harmful chemical substances in the environment is caused by binding to an intracellular receptor called an aromatic hydrocarbon receptor (hereinafter referred to as AhR) and its activity.

 [0003] In addition to dioxins, tobacco smoke contains many and complex aromatic hydrocarbons such as polybranched aromatic hydrocarbons such as cobrana PCB and benzpyrene. Has been suggested to play an important role in health hazards caused by tobacco smoke.

 [0004] Environmentally harmful chemical substances such as aromatic hydrocarbons cause various harmful effects in a very small amount. Therefore, the establishment of a method for detecting these toxic chemicals sensitively and quickly is an urgent issue in preventing health problems.

 [0005] Currently, high resolution gas chromatograph mass spectrometry is used for the detection and quantification of dioxins. Although this method is highly accurate, it requires a lot of labor and cost for pretreatment and measurement. Cost. In response to these current conditions, several simple measurement methods using cultured cells (bioassay methods such as the reporter bioassay method) have been reported in recent years.

[0006] In 2000, the US Environmental Protection Agency adopted this method as a screening method for environmental samples, and in 2002, screening for dioxins content in food and feed was conducted in the EU. The same method was certified as a law. Considering the current situation and the problems of the official method mentioned above, the Ministry of the Environment in Japan approved the application of the simplified measurement method in the dioxin contamination monitoring survey in 2004, and also the simplified measurement method of dioxins using the bioassay method. Is under consideration.

 [0007] In view of the current situation, the inventor has developed a simple and highly sensitive bioassay DRESSA method for detecting aromatic hydrocarbons in the environment using AhR activity as an index (Non-patent Document 1). This method is a new assembly method that uses a binding base sequence of AhR called dioxin response element (DRE) as a sensor and binds the SEAP gene downstream of it as a reporter.

 [0008] This gene structure was introduced into mouse hepatoma cells to establish HeDS cells 1 that express and secrete SEAP when AhR is activated by aromatic hydrocarbons. When a sample containing dioxin is added to the sensor cells, SEAP is secreted into the culture medium in a concentration-dependent manner. Therefore, by measuring the SEAP activity in the culture medium, it is possible to detect and quantify dioxins and polycyclic aromatic hydrocarbons in the sample.

 [0009] By this DRESSA method, it is possible to access a large number of environmental samples on a very small scale at low cost, in a short time, and with high sensitivity. It has also been confirmed that this system reacts sensitively to aromatic hydrocarbons in tobacco smoke (Non-patent Document 3).

 [0010] However, the Atsy system using cultured cells has some limitations. That is, 1) In order to add environmental samples to the culture system, extraction and concentration of environmental hazardous chemicals involves an artificial manipulation process, making direct evaluation of untreated samples difficult. 2) The effects of harmful chemicals on individual animals cannot be directly examined in the same exposure mode as in reality, 3) Combined harmful effects through multiple routes such as air 'food' water Inability to comprehensively assess exposure to chemicals, 4) Inability to study chronic long-term exposure effects.

[0011] In order to overcome these problems, it is necessary to establish a transgenic animal that expresses a marker gene when exposed to an aromatic hydrocarbon and use it for evaluation. To date, an example using a transgenic mouse in which a β-galatatosidase gene is incorporated downstream of DRE has been reported (Non-patent Document 2). [0012] The critical problems with this system are: 1) the effects of endogenous 13-galatatosidase cannot be ruled out and poorly quantified; 2) the marker protein is not secreted and the mouse must be killed during the measurement 3) Therefore, the same animal cannot be reused (that is, continuous evaluation using the same individual cannot be performed), and it is very expensive when used for environmental monitoring. 4) For the assembly For example, an operation of extracting an organ, crushing and dissolving a tissue, and extracting a marker tongue is necessary, which is complicated and time-consuming. These issues make it extremely difficult to actually use these mice for environmental monitoring. In addition, there has never been a transgenic ribota-animanole that expresses and secretes secreted reporter proteins.

 Non-patent document 1: Japanese Patent Application 2004-153293

 Non-patent literature 2: Willey JJ, btnppBR, BaggsRB, and GasiewiczTA. Aryl hydrocarbon re ceptor activation in genital tubercle, palate, and other embryonic tissues in 2,3,7, 8 Appl Pharmacol lb 1:33 -44, 1998.

 Non-Patent Document 3: PCT / JP2005 / 8875

 Disclosure of the invention

 Problems to be solved by the invention

 [0013] Therefore, the present invention can easily detect, for example, endocrine disrupting substances and some carcinogenic substances that are harmful chemical substances directly in the real environment, and can be a complex environmental harmful chemical substance. It provides a transgenic non-human mammal that enables comprehensive and continuous assessment of effects, and a method for monitoring environmental hazardous chemicals using the same.

 Means for solving the problem

[0014] The present invention is a transgenic non-human mammal that produces a marker protein and secretes it into a body fluid when placed in an environment containing harmful chemical substances. The environmentally hazardous chemicals are dioxins and Z or polycyclic aromatic hydrocarbons. The marker protein is preferably secretory alkaline phosphatase. By using secretory alkaline phosphatase as a marker protein, only a small amount of body fluid (eg blood) can be collected. Evaluation is possible, and it is not necessary to extract the organ by removing the organ and crushing and dissolving the tissue.

 [0015] The transgene is preferably a gene (MMTV-DRE-SEAP) in which a secreted alkaline phosphatase (SEAP) gene is linked downstream of a dioxins hypersensitive gene (MMTV-DRE). The non-human mammal is preferably a mouse or a rat.

[0016] The present invention allows a gene-introduced non-human mammal that produces a marker protein when placed in an environment containing a harmful chemical substance to act in the evaluation environment and is contained in the body fluid of the gene-introduced non-human mammal. And detecting and quantifying environmental harmful chemical substances contained in the evaluation environment using the marker protein activity as an index.

 [0017] The harmful chemical substances in the environment are dioxins and Z or polycyclic aromatic hydrocarbons, and the marker protein is preferably secretory alkaline phosphatase.

 [0018] In the present invention, a gene-introduced non-human mammal that produces a marker protein when placed in an environment containing harmful chemical substances is activated in contaminated air mainly composed of tobacco smoke, and the gene-introduced This method evaluates the biological toxicity of tapaco smoke using the activity of a marker protein contained in the body fluid of a human mammal as an index. It is preferable that the marker protein is a secreted Al force phosphatase.

 [0019] The present invention is a monitoring method for environmental hazardous chemical substances, which performs the above-mentioned environmental hazardous chemical measurement method in time series and monitors environmental hazardous chemical substances contained in an evaluation environment.

 [0020] The present invention is a screening method for an accelerating substance that suppresses or suppresses an aromatic hydrocarbon receptor function using the above-described transgenic non-human mammal.

 [0021] Further, the present invention provides an aromatic hydrocarbon receptor comprising, as an active ingredient, a substance obtained by screening a promoter that promotes or suppresses the function of the above-described aromatic hydrocarbon receptor. It is a substance that promotes or suppresses function.

 The invention's effect

[0022] According to the present invention, a hazardous chemical substance in the environment directly in an actual environment, for example, Secretory disruptors and some carcinogens can be easily detected. In addition, the effects of complex environmental hazardous chemicals can be evaluated in an integrated manner.

 Brief Description of Drawings

 FIG. 1 is a structural diagram of a dioxin-like responsive plasmid (pDRE-SEAP).

 FIG. 2 shows an electrophoretic image of purified DNA fragment MMTV-DRE-SEAP-polyA.

 [Fig. 3] Diagram showing the integration of the SEAP gene into the offspring genome (PCR method).

 FIG. 4 is a graph showing serum SEAP activity before oral administration of dioxin and 24 hours after administration.

 FIG. 5 is a graph showing changes in blood SEAP activity over time.

 FIG. 6 is a graph showing changes in blood SEAP activity when the oral dose of TCDD is changed.

 FIG. 7 shows the results of observation of gene expression of SEAP, cytochro meP450 (CYPlAl), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the liver before and after oral administration of TCDD on the 3rd and 21st day by PCR.

 FIG. 8 is a diagram showing sex differences in the reactivity of DRESSA mice to TCDD.

 [Fig. 9] A graph showing the transition of blood SEAP activity after a DRESSA mouse was temporarily exposed to cigarette smoke.

 Explanation of symbols

[0024] 1 HeDS cells

 10 Dioxins-responsive plasmid (pDRE-SEAP)

 11 Dioxins high sensitivity gene sequence (MMTV-DRE)

 12 Secreted marker protein gene (SEAP)

 13 Poly address signal (polyA)

 BEST MODE FOR CARRYING OUT THE INVENTION

[0025] The best mode for carrying out the invention will be described below.

[0026] FIG. 1 shows a structural diagram of dioxins and Z or polycyclic aromatic hydrocarbon reactive plasmid PDRE-SE AP10. pDRE-SEAPIO incorporates a dioxin-responsive DNA sequence (DRE) into a part of the mouse breast cancer virus promoter (MMTV). Dioxins highly sensitive DNA sequence (hereinafter referred to as MMTV-DRE11) and secretory alkaline phosphatase (hereinafter referred to as SEAP) gene 12 located downstream thereof, and polyadenylation signal derived from simian virus 40 (hereinafter referred to as SV40) , PolyA13).

 [0027] MMTV-DRE11 is known to be activated in response to dioxins, and is one in which four DREs are inserted into a part of the MMTV promoter. Responsive to dioxins compared to DRE alone. SEAP was used as the marker protein. A bioaccessory system for dioxins using secreted marker proteins, including SEAP, has not been established. SEAP is a marker protein that can be detected with high sensitivity along with luciferase. Unlike luciferase, SEAP is secreted outside the cell, so it is not necessary to destroy the cells and extract the protein.

 [0028] pDRE-SEAPIO is prepared by a conventional genetic engineering method. That is, the MMTV-DRE11 fragment excised and purified with restriction enzymes should have no promoter! Insert T4 DNA ligase upstream of SEAP gene 12 of the SEAP plasmid!

 [0029] T4 DNA ligase is an enzyme that links the 5 'and 3' ends of adjacent DNA strands. The prepared pDRE-SEAPIO is introduced into E. coli and increased in quantity, and pDRE-SEAP10 is purified by genetic engineering routine methods.

 [0030] This pDRE-SEAP is cleaved with restriction enzymes Mlul and Sail, and the 3.8 kb DNA fragment MMTV-DRE-SEAP-polyA is purified by electrophoresis using agarose gel. This is injected into the male pronucleus of the fertilized egg collected from the pregnant mouse (C57BL / 6) force and transferred to the fallopian tube of a pseudopregnant mouse. The transgenic mouse of the present invention can be prepared by obtaining a litter from the transplanted fertilized egg.

 Example

 [0031] The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto.

[0032] pDRE-SEAPIO was prepared by a conventional genetic engineering method. The pDRE-SEAP was cleaved with restriction enzymes Mlul and Sail, and the 3.8 kb DNA fragment MMTV-DRE-SEAP-polyA was purified by electrophoresis using agarose gel. Figure 2 shows the purified DNA fragment MMTV-DRE-SE It is an electrophoretic image of AP-polyA. The DNA fragment MMTV-DRE-SEAP-polyA was injected into the male pronucleus of fertilized ovum collected from pregnant mice (C 57BL / 6), and transplanted into the oviduct of pseudopregnant mice to obtain pups. The baby was successfully weaned. A part of the tail of the offspring was collected, DNA was extracted, and PCR was performed, so that 5 offspring (No.2,5,15,24,25) Incorporation was confirmed (Figure 3). Of these mice, No. 25 was mated with a wild-type mouse, and the resulting male offspring whose SEAP gene integration was confirmed by PCR were used in the experiments described below. .

 [0033] Blood of isolated transgenic mice (DRESSA mice) and wild-type mice (C57BL / 6) was collected from the tail vein and then 2,3,7,8-tetrachlorodibenzo-p using a gastric sonde. -di oxin (TCDD; dioxin) was orally administered by gavage at 5 ng / g body weight. Thereafter, blood samples were collected every 24 hours, and the SEAP activity in the serum was measured with a fluorescence system.

 [0034] Specifically, 5 µl of serum was supplemented with 15 µl buffer and incubated at 65 ° C for 30 minutes to inactivate endogenous alkaline phosphatase activity. In addition, a buffer containing L-homoarginine, an inhibitor of endogenous alkaline phosphatase, was allowed to stand for 201 minutes for 5 minutes, and then 15 μl of substrate CSPD was added. After leaving it in the dark for 30 minutes, use a luminometer to establish SEAP activity.

 [0035] As shown in FIG. 4, when TCDD was administered to DRESSA mice, blood SEAP activity reached about 12 times that before TCDD administration after 24 hours. On the other hand, when TCDD was administered to wild-type mice, no increase in blood SEAP activity was observed.

 FIG. 5 is a graph showing changes over time in blood SEAP activity thereafter. After a single oral dose of TCDD, the blood SEAP activity continued to increase and peaked at 28 days, and then gradually decreased (change indicated by 參). On the other hand, no increase in blood SEAP activity was observed in DRESSA mice not treated with TCDD (changes indicated by circles).

 [0037] As described above, by using DRESSA mice, it is possible to monitor environmental hazardous chemicals using SEAP activity in serum as an index. In addition, the blood SEAP activity of DRESSA mice when unstimulated was about 2.6 times that of wild-type mice.

[0038] FIG. 6 shows changes in blood SEAP activity when the dose of TCDD is changed. TCDD was gavaged at 0.5, 1, 5 ng / g body weight, and blood SEAP activity was measured until day 21 It is a graph which shows the result. As shown in the graph of Fig. 6 (a), blood SEAP activity increased depending on the dose of TCDD. As shown in the graph of Fig. 6 (b), even at a low dose of 0.5 ng / g body weight, a transient increase in SEAP activity peaked on day 7 was observed (about 4 times). In other words, it can be seen that blood SEAP activity in DR ESSA mice increases in relation to the exposure to harmful chemicals. At the dose of 0.1 ng / g body weight, no significant increase in SEAP was observed.

 [0039] Fig. 7 shows the PCR analysis of the gene expression of SEAP, cytochrome P450 (CYP1A1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the liver before and after TCDD administration and on day 21 and day 21. It is. CYP1A1 is an enzyme known to be induced by TCDD, and GAPDH is an enzyme that is not affected by its expression ability TCDD. Although SEAP gene expression was not observed in the liver of DRESSA mice before TC DD administration, induction of SEAP gene expression was observed after TCD D administration, and the expression level was higher on day 21 than on day 3. Indicated. This process correlates very well with the transition of blood SEAP activity shown in FIG. This means that the blood SEAP activity reflects the degree of DRE activity at any given time!

 [0040] FIG. 8 shows gender differences in the reactivity of DRESSA mice to TCDD. DRESSA No. 25 was mated with a wild-type mouse, and 5 ng / g body weight of TCDD was forcibly administered orally to the obtained male and female mice, and then SEAP activity in blood was measured until day 21. As a result, male DRESSA mice should be used for monitoring harmful chemicals that are less reactive than males that have a significant increase in blood SEAP activity in females. It was revealed.

[0041] Figure 9 shows that tobacco smoke is known to contain many types of AhR-active substances. It is a graph following the transition of medium SEAP activity. Specifically, mice were placed in 500 ml bottles and exposed 12 times every 15 minutes to 45 ml of mainstream smoke of 10 mg tar. In the meantime, a net was put on the opening of the bottle so that the mouse could inhale fresh air freely (transition indicated by 參). As a control group, DRESSA mice were placed in bottles in the same manner and exposed to 45 ml of air 12 times every 15 minutes (changes indicated by circles). After exposure to cigarette smoke, mice are kept normally After returning to the environment, blood was collected every day. As shown in Fig. 9, after exposure to cigarette smoke, blood SEAP activity increased significantly at 12 hours, peaked at 24 hours, and then gradually decreased until 4 days after exposure. An increase was observed. In other words, it is possible to directly monitor the contamination of the outside air and room air with the DRESSA mouse.

[0042] This specification is based on Japanese Patent Application No. 2005-194184 filed on Jul. 1, 2005. All this content is included here.

 Industrial applicability

 [0043] Non-transgenic gene that produces and secretes SEAP through the activation of aromatic hydrocarbon receptor (AhR) in response to harmful chemical substances such as aromatic hydrocarbons including dioxins of the present invention If human mammals are used, organisms are given specific outside air in incinerators or factories where smoke is a problem, along highways where exhaust gases are a problem, or tobacco smoke in smoking rooms or smoking vehicles. Continuous monitoring of impacts is possible.

 [0044] In addition, monitoring of harmful substances by directly ingesting 'drinking food and drinking water', drinking environmental samples such as river water, and extracting harmful chemical substances extracted from soil force, or activating AhR It is possible to search for chemical substances such as synthetic compounds and natural product extracts, and to apply the results to preventive medicine.

 [0045] In addition, the search for substances that suppress the adverse effects of AhR activation, such as synthetic compounds and natural product extracts, and the application of the results to therapeutic medicine, as well as the quantification of the impact of tobacco brands on health Enable the development of new systems and indicators

Claims

The scope of the claims  [I] Transgenic non-human mammals that produce marker proteins when placed in an environment containing hazardous chemicals.  [2] The transgenic non-human mammal according to claim 1, wherein the harmful chemical substance is dioxins and Z or a polycyclic aromatic hydrocarbon. [3] The marker protein according to claim 1 or claim 2, wherein the marker protein is secretory alkaline phosphatase. 2. The transgenic non-human mammal according to 2. [4] The transgene is a gene (MMTV-DRE-SEAP) in which a secreted alkaline phosphatase (SEAP) gene is linked downstream of a dioxins hypersensitive gene (MMTV-DRE). The transgenic non-human mammal according to claim 1. 5. The gene-introduced non-human mammal according to claim 1, wherein the non-human mammal is a mouse or a rat.  [6] A transgenic non-human mammal that produces a marker protein when placed in an environment containing harmful chemical substances is activated in the evaluation environment, and the marker protein contained in the body fluid of the transgenic non-human mammal is A method for quantifying hazardous chemical substances in the environment, wherein activity is used as an index to quantify harmful chemical substances contained in the evaluation environment.  7. The method for quantifying environmental hazardous chemical substances according to claim 6, wherein the hazardous chemical substances are dioxins and Z or polycyclic aromatic hydrocarbons.  [8] The method for quantifying an environmental harmful chemical substance according to [6] or [7], wherein the marker protein is secretory alkaline phosphatase.  [9] Transgenic non-human mammals that produce marker proteins when placed in an environment containing harmful chemical substances are activated in contaminated air mainly composed of tobacco smoke, and are introduced into the body fluid of the transgenic non-human mammals. A method for evaluating the biological toxicity of tobacco smoke using the activity of the marker protein contained as an index to evaluate the biological toxicity of tobacco smoke. 10. The method for evaluating the biological toxicity of tobacco smoke according to claim 9, wherein the marker protein is secretory alkaline phosphatase. [II] The transgene is a gene (MMTV-DRE-SEAP) in which a secreted alkaline phosphatase (SEAP) gene is linked downstream of a dioxin-sensitive gene (MMTV-DRE) 11. The method for evaluating biological toxicity of tobacco smoke according to claim 9 or claim 10. [12] The method for evaluating biological toxicity of tobacco smoke, wherein the non-human mammal is a mouse or a rat. [13] A monitoring method for environmental hazardous chemicals, wherein the method for quantifying environmental hazardous chemicals according to claim 6 or claim 7 is performed in a time series to monitor the hazardous chemicals contained in the evaluation environment.  [14] Screening of a promoter that promotes or suppresses the function of an aromatic hydrocarbon receptor using the transgenic non-human mammal according to any one of claims 1 to 3. Method.  [15] A substance that promotes or suppresses the function of an aromatic hydrocarbon receptor, comprising as an active ingredient the substance obtained by the screening method according to claim 14.