JP2006014716A - Method for searching anti-alkylating substance, and anti-alkylating agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for searching an anti-alkylating substance that inhibits/repairs alkylation inducing O<SP>6</SP>methylguanine among alkylations, and to obtain anti an alkylating agent searched by the method. <P>SOLUTION: The method for searching the anti-alkylating agent that inhibits alkylation levels comprises measuring the expression level of fused gene having an alkylation-repairing gene that recognizes O<SP>6</SP>methylguanine and adding a substance to be tested to an alkylated DNA of a host cell induced by the addition of an alkylating agent. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for searching for anti-alkylating substances.

  In recent years, malignant neoplasms (hereinafter referred to as cancer) are the most common causes of death among Japanese. Although the cause of cancer has not yet been fully elucidated, DNA damage is one cause of its canceration. DNA damage refers to physical or chemical changes that occur in DNA due to environmental factors. Among them, DNA alkylation, which is one of the chemical changes, has recently attracted attention.

Alkylation of DNA usually means that a base on DNA is alkylated. In particular, O ⁶ methylguanine in which the oxygen atom at the 6-position of guanine base is alkylated is said to be involved in canceration. Yes.

For example, when O ⁶ methylguanine is produced on E. coli DNA, transcription of the ada gene occurs and Ada protein is produced. This Ada protein is a protein that repairs O ⁶ methylguanine in DNA, and has a correlation with the amount of O ⁶ methylguanine on DNA generated in cells.

By measuring the amount of O ⁶ methylguanine produced, it becomes possible to confirm alkylation related to canceration of DNA. Here, as a measuring method, there is a method using a transformant encoding a fusion protein of Ada protein and β-galactosidase (Non-patent Document 1).

According to this measurement method, it is possible to perform measurement specialized for O ⁶ methylguanine in alkylation of DNA without using a large-scale measurement method by expensive equipment such as HPLC, GCMS or LCMS and technology. It is.

  Thus, the DNA alkylation mechanism has been studied in various ways in recent years.

  As the alkylation mechanism is elucidated, research on the existence of a substance that suppresses this alkylation, that is, an “anti-alkylating substance” is beginning.

However, for example, in the aforementioned measuring method, to measure the O ⁶ methyl guanine amount, it is possible to search the alkylating agent according to the O ⁶ methyl guanine, it is impossible to search for the anti-alkylating agent On the contrary, a method for searching for an anti-alkylating substance that suppresses canceration has not been established yet.

In addition, in order to search for an anti-alkylating substance, it is necessary to measure various test substances in large quantities at once and stably with high accuracy. In the above-described measurement method, the amount of O ⁶ methylguanine is measured. Even if it is possible, it is difficult to measure a large amount of samples at a time with stability and accuracy.
(Y. Nakabepupu Mutation Research 146 (1985) 155-167)

The present invention has been made in view of the above circumstances, and a method for searching for an anti-alkylating substance that suppresses and repairs alkylation that causes O ⁶ methylguanine in alkylation, and an anti-alkylation searched by the searching method. The purpose is to obtain an agent.

Means for Solving the Invention

  In order to achieve the above object, the present invention provides the following means.

(1) A method for searching for an anti-alkylating substance, comprising the steps of: adding an alkylating agent to a host cell; and adding a test substance to the host cell.
(2) The method for searching for an anti-alkylating substance according to (1), wherein the host cell has a fusion gene of an alkylation repair gene and a gene serving as an index,
(3) The method for searching for an anti-alkylating substance according to (1) or (2), wherein the host cell is derived from E. coli, and (4) the alkylation repair gene is an E. coli ada gene. (1) to (4), wherein the method for searching for an anti-alkylating substance according to (1) to (3) is characterized in that the gene serving as the indicator is Escherichia coli lacZ. (6) The method for searching for an anti-alkylating substance according to (1) to (5), wherein the host cell DNA is alkylated in the step of adding the alkylating agent. (7) The method for searching a substance, (7) The oxygen atom at the 6-position of the guanine base on the DNA is alkylated in the step of adding the alkylating agent. Search method for anti-alkylating substance, (8 The alkylating agent is MNNG (1-methyl-3-nitro-1-nitrosoguanidine; 1-Methyl-3-nitro-1-nitrosoguanidine), MNU (N-nitroso-N-methylurea; N-Nitroso-N- (9) the method for searching for an anti-alkylating substance according to any one of (1) to (7), which is methylure), DEN (N-nitrosodiethylamine) or ENU (N-nitroso-N-ethylurea); The method for searching for an anti-alkylating substance according to any one of (1) to (8), wherein the alkylation of the DNA is suppressed when the test substance is added before adding the alkylating agent, (10) When adding the test substance after adding the alkylating agent, alkylating the DNA (1) The method for searching for an anti-alkylating substance according to (1) to (8), wherein the method comprises (11) a step of adding a test substance to a host cell that is naturally alkylated And (12) the host cell is derived from Escherichia coli, wherein the host cell has a fusion gene of an alkylation repair gene and an indicator gene. (11) The method for searching for an anti-alkylating substance according to (11), (13) The anti-alkylating substance according to (11) or (12), wherein the alkylation repair gene is an E. coli ada gene (14) The method for searching for an anti-alkylating substance according to (11) to (13), wherein the index gene is Escherichia coli lacZ, and (15) guanine on the DNA The method for searching for an anti-alkylating substance according to any one of (11) to (14), wherein the oxygen atom at the 6-position of the base is naturally alkylated, (16) Addition of an alkylating agent to the host cell And a method for searching for an anti-alkylating substance comprising a step of adding a test substance to the host cell, or a step of adding a test substance to a host cell that is naturally alkylated. A method for searching for an anti-alkylating substance, wherein the host cell is derived from Escherichia coli and has been searched by a method for searching for an anti-alkylating substance having a fusion gene of an alkylation repair gene and an indicator gene. (17) Captopurine, chitosan, chlorophyllin, L-citrulline, curcumin, fisetin, L-glutathione, phenethylisothiocyanate Quercetin, ascorbic acid, rutin, Shisuchiamin, epicatechin, beta-carotene, anti alkylating agent characterized in that it contains chitosan and / or Roman phloroglucinol.

  Conventionally, it has been known that the expression of the ada gene is increased by several alkylating agents that cause alkylation of DNA that contributes to canceration. However, no attempt has been made to search for anti-alkylating substances.

  Therefore, the present inventors not only search for an alkylating substance, but can suppress the DNA alkylation reaction when the alkylating substance is taken into the body, or can occur spontaneously in the body. It came to establish the method of searching for an anti-alkylation substance as a test substance, such as suppressing and repairing alkylation. Furthermore, in order to obtain an efficient and reliable result, a high-throughput system capable of measuring a large amount of samples at a time has been established.

  Hereinafter, embodiments of the present invention will be described.

  (First embodiment)

  A method for searching for an anti-alkylating substance when an alkylating agent is added to the host cell will be described.

  The method for searching for an anti-alkylating substance of the present invention (hereinafter referred to as “search method of the present invention”) comprises a step of adding an alkylating agent to a host cell and a step of adding a test substance to the host cell. It is characterized by that.

  That is, in the search method of the present invention, an alkylating agent is added to a host cell to alkylate the host cell DNA, and at the same time, a test substance is added to the host cell, whereby an alkylated base on the DNA is obtained. Searching for a substance that suppresses the amount (hereinafter referred to as the alkylation amount), that is, an anti-alkylation substance.

  A host cell refers to a cell that takes in and propagates a target gene or the like. Here, for example, E. coli is used. As other host cells, Bacillus subtilis, yeast, nematodes, fly / mouse / human cell lines and the like may be used.

Here, the alkylation of DNA means that the oxygen atom at the 6-position of the guanine base on the DNA is alkylated, that is, O ⁶ methylguanine is formed.

  In addition, the host cell used in the present invention, for example, E. coli, is characterized in that a fusion gene comprising an alkylation repair gene and an indicator gene is incorporated as a target gene.

  The alkylation repair gene in this case is a gene that recognizes the alkylation of DNA and encodes a protein that repairs the alkylation, and is a gene that is expressed with the amount of DNA alkylation.

  When the amount of alkylation of E. coli DNA is large, the alkylation repair gene is expressed in a large amount. Conversely, when the amount of DNA alkylation in the host cell is small, the alkylation repair gene is expressed in a small amount.

  Therefore, to measure the alkylation amount of Escherichia coli DNA, the expression level of the alkylation repair gene may be measured.

  However, since it is difficult to measure the expression level of the alkylation repair gene alone, an alkylation repair gene is prepared by preparing a fusion protein fused with the index gene and measuring the index gene amount. Can be measured. That is, the measured expression level of the alkylation repair gene is the DNA alkylation level.

Here, the alkylation repair gene is preferably an ada gene (a gene encoding O ⁶ methylguanine DNA-methyltransferase).
The ada gene is a gene that recognizes and repairs O ⁶ methylguanine, and its expression increases in proportion to the amount of DNA alkylation. Therefore, the production amount of the alkylated base of the DNA according to the present invention is measured. In some cases. A gene other than the ada gene may be used as long as it encodes a protein that recognizes O ⁶ methylguanine and increases its expression in proportion thereto.

  Further, the gene serving as an index preferably refers to a reporter gene. More preferably, among the reporter genes, lacZ (gene encoding β-galactosidase) is preferable.

  A reporter gene is a gene that allows easy confirmation of at what time and to what extent a recombinant gene produced by recombinant DNA technology was expressed. In addition, lacZ is well known as a gene that serves as an index by fusing with a target gene whose expression level is to be measured, ie, a reporter gene, that maintains its enzymatic activity after fusion, its enzymatic reaction, etc. Since it is shown that color develops due to decomposition of the substrate, it is preferable. As long as it is a reporter gene, a gene other than lacZ, such as luciferase, may be used.

Here, examples of the alkylating agent include MNNG (1-methyl-3-nitro-1-nitrosoguanidine). Since MNNG produces a large amount of O ⁶ methylguanine even in the alkylating agent, the expression of the ada gene is strongly induced, so whether or not the alkylation of DNA is suppressed when the anti-alkylating agent is added. It is easy to make a determination. That is, the alkylation by MNNG is due to the large proportion of O ⁶ methylguanine produced in the base on the alkylated DNA.
As other alkylating agents, MNU (N-nitroso-N-methylurea), DEN (N-nitrosodiethylamine), ENU (N-nitroso-N-ethylurea), etc., in which O ⁶ methylguanine is produced in a large proportion But you can.

  In addition, the anti-alkylating substance may be added to the host cell before or after the addition of the alkylating substance. Thus, before adding an alkylating substance, for example, before adding an alkylating substance, by measuring the amount of DNA alkylated before adding the alkylating agent to the host cell or after adding the alkylating substance, If an anti-alkylating substance is added to suppress alkylation, it can be assumed that the anti-alkylating substance suppresses alkylation of DNA. This makes it possible to optimize the intake time when the anti-alkylated substance searched for by the search method in the future is applied to, for example, foods and drugs.

  From the above configuration, the method for searching for an anti-alkylating substance of the present invention is caused by adding an alkylating agent by measuring the amount of a fusion gene having an alkylation repair gene in a host cell and further adding a test substance. It is possible to search for anti-alkylating substances that suppress the alkylation amount of the host cell DNA.

  (Second Embodiment)

  Next, a method for searching for an anti-alkylating substance that suppresses naturally occurring alkylation of host cell DNA will be described.

  The search method of the present invention comprises a step of adding a test substance to a host cell, wherein the host cell is derived from Escherichia coli and has a fusion gene of an alkylation repair gene and a gene serving as an index. To do.

  That is, unlike the first embodiment, the search method of the present invention differs from the first embodiment in that no alkylating agent is added to the host cell, that is, a substance that suppresses the spontaneous alkylation of host cell DNA, that is, anti-alkylation. Search for a substance.

  Here, spontaneous alkylation refers to DNA alkylation that can occur naturally even when a substance that causes alkylation such as an alkylating agent is not added to the host cell. For example, naturally occurring alkylation includes those occurring in the course of substance metabolism.

  In order to measure the amount of alkylation by spontaneous alkylation of DNA, it is preferable to measure the amount of alkylation over time.

  That is, if the amount of alkylation is measured over time, it can be confirmed that alkylation has occurred.

  In the method of the present invention, it is possible to search for an anti-alkylating substance that suppresses alkylation by adding a test substance to a host cell in which this spontaneous alkylation has occurred.

  In other words, by adding an anti-alkylating substance over time to such spontaneous alkylation, it is effectively suppressed at any point against spontaneous alkylation. You can see if you can. Therefore, the anti-alkylating substance searched for by the searching method of the present invention can be used in the field of cancer prevention / treatment.

  From the above configuration, the method for searching for an anti-alkylating substance of the present invention can search for an anti-alkylating substance against spontaneous DNA alkylation.

  The anti-alkylating substance of the present invention obtained as described above can be used, for example, as food, medicine and the like. When used as a food, it may be added to existing food as well as the anti-alkylating substance of the present invention alone. Moreover, when using as a pharmaceutical, it can formulate using the carrier used normally and can adjust it to dosage forms, such as injection, oral, and transdermal. For example, when used for humans, the anti-alkylating substance may be appropriately selected from about 1 μg to 100 mg / kg from once to several times a day.

  Hereinafter, the present invention will be described in detail using examples. However, it goes without saying that the present invention is not limited to this.

  <Detection of anti-alkylation activity>

In the following, the antialkylation activity was measured when the test substance and the alkylating agent were added almost simultaneously or after the test substance was added. In addition, as a test substance of this example, an antimutagenic substance was used, and antialkylation activity was measured.
Strain E. coli; YN113 W3110 trp ^R lacZ ValR azi TrpE929 (am) trpA9761 (am) :: pMY3
LB medium (500 ml): 5 g tryptone (DIFCO)
Yeast Extract (DIFCO) 2.5g
NaCl (Wako) 5g
Z buffer (500 ml): 0.2 M Na ₂ HPO ₄ (Wako) 150 ml
0.2M NaH ₂ PO ₄ (Wako) 100ml
KCl (Wako) 0.375g
MgSO ₄ · 2H ₂ O (Wako) 0.123 g
β-mercaptoethanol (sigma) 0.35 ml

  (1) Preculture 1: Using a large test tube, the cells were cultured at 37 ° C. for 14 hours in 2.5 ml of LB medium (final concentration: 50 μg / ml ampicillin sodium salt).

  (2) Preculture 2: Using a 200 ml Erlenmeyer flask, preculture 1 was diluted 1/100 with 30 ml of LB medium (final concentration 50 μg / ml ampicillin sodium salt) and cultured at 37 ° C. for 1 hour 50 minutes. did.

  (3) The alkylating agent MNNG final concentration of 0.005 μg / ml was added to (2).

  (4) The final concentration of PEITC (phenethyl isothiocyanate (Sigma)) known as an antimutagenic substance is 0.5, 1, 2, 5, 10, 25 μg / ml as a test substance. Diluted sample was used.

  (5) Sample dispensing. 1 ml or 1.5 ml of (3) was dispensed to the small examiner of (4) and stirred. Samples of each tube were dispensed into 2 ml 96-well plates at 250 μl per well and cultured (4 or 5 wells were dispensed for each small test tube (concentration)).

(6) Sampling and sample adjustment. After adding the alkylating agent, a sample was collected from each hole of the plate 1 hour after the start of culture.
A 0.5 ml 96-well plate was prepared, and 150 μl of Z buffer was put in advance. Further, 30 μl of each culture sample was added, one drop of toluene was added, and then the lid was capped and vortexed. At the same time, 100 μl of the sample was collected in a 96-well assay plate, and the absorbance at a wavelength of 600 nm was measured with a plate reader to measure the turbidity of the sample.

(7) Assay. After all sampling is complete, centrifuge briefly and open the lid. Incubate at 37 ° C. for 30 minutes to volatilize toluene. Dispense 50 μl assay buffer (1 μg / ml final concentration of ONPG in Z buffer (Sigma)) per 96-well assay plate per sample. Add 30 μl of sample and leave at 28 ° C. for 10 minutes. The reaction is stopped by adding 40 μl of 1M Na ₂ CO ₃ . Absorbance at a wavelength of 414 nm and a wavelength of 550 nm was measured with a plate reader.

(8) Activity was calculated | required by the following calculation.
(Anti-alkylation activity) U (unit) = 1000 × (A ₄₁₄ -1.75 × A ₅₅₀ ) / (t × 0.0075 × A ₆₀₀ )
t; time of incubation at 28 ° C. (minutes)
A ₄₁₄ ; absorbance at a wavelength of 414 nm A ₅₅₀ ; absorbance at a wavelength of 550 nm A ₆₀₀ ; absorbance at a wavelength of 600 nm

  (9) Data processing. In addition to the comparison of the number of units, the influence of the test substance in each sample when the control was 1 was detected.

<Result>
It was shown that the expression of the ada gene by the alkylating agent MNNG is suppressed in a concentration-dependent manner. The concentration-dependent antialkylation activity of the test substance PEITC was detected at 1 and 2 hours when strong expression was induced by addition of the alkylating agent MNNG. FIG. 1 shows the antialkylation activity of the alkylating agent MNNG and the test substance PEITC at 1 hour. The vertical axis shows the ratio of inhibition when the test substance is added when the control 1 is not added with the test substance. The horizontal axis represents the concentration of the test substance.

  In addition to PEITC, Captopril (captopril (Wako)), Chitosan (chitosan (Sigma)), Chlorophyllin (chlorophyllin (Sigma)), L-citrulline (L-citrulline (Sigma)), curcumin (curcumin) (Wako)), Fisetin (Fisetin (Sigma)), L-Glutathione (L-glutathione), and Quercetin (quercetin (Sigma)) were also confirmed to have anti-alkylating activity.

<Detection of anti-alkylation activity>
The anti-alkylation activity when the test substance was added before adding the alkylating agent was measured below.
Strain E. coli; YN113 W3110 trp ^R lacZ ValR azi TrpE929 (am) trpA9761 (am) :: pMY3

  (1) Pre-culture 1 Using a large test tube, the cells were cultured in 2.5 ml of LB medium (final concentration 50 μg / ml ampicillin sodium salt) at 37 ° C. for 14 hours.

  (2) Preculture 2 (1) was diluted 1/100 with 30 ml of LB medium (final concentration 50 μg / ml ampicillin sodium salt).

  (3) The test substance was diluted so that the final concentration of β-carotene (β-carotene (Sigma)) would be 1, 5, 10, 25 μg / ml, and placed in a large test tube.

  (4) 1.5 ml of (2) was added to (3), and cultured with shaking at 37 ° C. for 1 hour and 50 minutes.

  (5) The alkylating agent MNNG (final concentration 0.005 μg / ml) was added to the small tester of (4) and stirred.

  (6) A sample of each tube was dispensed into a 2 ml 96-well plate at 250 μl per well (4 wells were dispensed for each small test tube (concentration)).

  (7) Sampling and sample adjustment. Samples were collected from each well of the plate at 1 or 2 hours after addition of the alkylating agent MNNG. A 0.5 ml 96-well plate was prepared, and 150 μl of Z buffer was put in advance. Further, 30 μl of each culture sample was added, one drop of toluene was added, and then the lid was capped and vortexed. At the same time, 100 μl of the sample was collected in a 96-well assay plate, and the absorbance at a wavelength of 600 nm was measured with a plate reader to measure the turbidity of the sample.

(8) Assay. After all sampling was completed, the tube was lightly centrifuged and the lid was opened. The mixture was incubated at 37 ° C. for 30 minutes to evaporate toluene. 50 μl of assay buffer (final concentration of ONPG in Z buffer: 1 μg / ml) per sample was dispensed into a 96-well assay plate. To this was added 30 μl of sample and left at 28 ° C. for 10 to 90 minutes. The reaction was stopped by adding 40 μl of 1M Na ₂ CO ₃ . Absorbance at a wavelength of 414 nm and a wavelength of 550 nm was measured with a plate reader.

(9) Activity was determined by the following calculation.
(Anti-alkylation activity) U (unit) = 1000 × (A ₄₁₄ -1.75 × A ₅₅₀ ) / (t × 0.0075 × A ₆₀₀ )
t; time of incubation at 28 ° C. (minutes)

  (10) Data processing. In addition to the comparison of the number of units, the influence of the test substance when the control was set to 1 was detected.

<Result>
In the case where the test substance is cultured in advance under the condition that the test substance is excessively present in the cell, in other words, when the test substance is cultured with the cell before the addition of the alkylating agent, the anti-antibody in Example 1 is used. Among substances whose alkylation activity has not been clearly confirmed, there is a substance whose expression suppression of the ada gene has been confirmed. Test substances include Captopril (captopril (Wako)), Ascorbic acid (ascorbic acid (vitamin C) (Sigma)), Rutin (rutin (vitamin P) (Sigma)), β-carotene (β-carotene (Sigma)) ) Was confirmed to suppress alkylation of the alkylating agent MNNG. FIG. 2 shows the antialkylation activity of pre in which the alkylating agent MNNG was added before the addition of the test substance β-carotene and the post in which the alkylating agent MNNG was added after the addition of the test substance. . The vertical axis represents the ratio of inhibition by adding the test substance, assuming that the control to which the test substance is not added is 1. The horizontal axis represents the concentration of the test substance.

<Search for substances that suppress spontaneous alkylation>
In the following, anti-alkylation activity for suppressing spontaneous alkylation, which is a case where no alkylating agent is added, was measured.

  (1) Pre-culture 1 Using a large test tube, the cells were cultured in 2.5 ml of LB medium (final concentration 50 μg / ml ampicillin sodium salt) at 37 ° C. for 14 hours.

  (2) Preculture 2 Using a 200 ml Erlenmeyer flask, the above was diluted 1/100 with 30 ml of LB medium (final concentration 50 μg / ml ampicillin sodium salt) and cultured at 37 ° C. for 1 hour 50 minutes.

  (3) Sample dispensing and main culture. 1 ml of (2) was dispensed to the large examiner and stirred. The culture was continued at 37 ° C.

  (4) The test substance is diluted so that the final concentration of PEITC, which is known as an antimutant substance, is 1, 5, 10, 25 μg / ml. The test substance was added. After stirring, the sample of each tube was dispensed into a 2 ml 96-well plate by 250 μl per well (the same sample of each test tube was dispensed into 5 holes).

  (5) Sampling and sample adjustment. Samples were collected from the large examiner at 1, 2, and 4 hours after the main culture. Samples were collected from the respective holes at 6, 7, and 8 hours. In advance, 150 μl of Z buffer was placed in each well of a 0.5 ml 96-well plate, 30 μl of each culture sample was added, one drop of toluene was added, and then the lid was capped and vortexed. At the same time, 100 μl of the sample was collected in a 96-well assay plate, and the absorbance at a wavelength of 600 nm was measured with a plate reader to measure the turbidity of the sample.

(6) Assay. After all sampling is complete, centrifuge briefly and open the lid. The mixture was incubated at 37 ° C. for 30 minutes to evaporate toluene. 50 μl of assay buffer (final concentration of ONPG in Z buffer: 1 μg / ml) per sample was dispensed into a 96-well assay plate. 30 μl of sample was added to this and left at 28 ° C. for 45 minutes. The reaction was stopped by adding 40 μl of 1M Na ₂ CO ₃ . Absorbance at a wavelength of 414 nm and a wavelength of 550 nm was measured with a plate reader.

(7) Activity was calculated | required by the following calculation.
(Anti-alkylation activity) U (unit) = 1000 × (A ₄₁₄ -1.75 × A ₅₅₀ ) / (t × 0.0075 × A ₆₀₀ )
t; time of incubation at 28 ° C. (minutes)

  (8) Data processing. In addition to the comparison of the number of units, the influence of the test substance in each sample when the control was 1 was detected.

<Result>
Β-carotene, Chitosan (chitosan (Sigma)), Chlorophyllin (chlorophyllin (Sigma)), and PEITC were confirmed as substances that suppress spontaneous alkylation when no alkylating agent was added. FIG. 3 shows the antialkylation activity over time at various concentrations of the test substance PEITC against naturally occurring alkylation. The vertical axis represents the ratio of inhibition by adding the test substance, assuming that the control to which the test substance is not added is 1. The horizontal axis represents the concentration of the test substance.

  <Detection of anti-alkylation activity when MNNG, MNU, ENU and DEN alkylating agents are used>

  In the following, the antialkylation activity was measured when the alkylating agents MNU, ENU and DEN were added in addition to the alkylating agent MNNG.

  As in Example 1, MNNG (0.05 μg / ml), MNU (50 μg / ml), ENU (1 μg / ml), DEN (100 μg / ml) were used as the alkylating substance, and PEITC 2 μg / ml was used as the test substance. Used to measure antialkylation activity.

  FIG. 4 shows a comparison of the antialkylation activity with the test substance PEITC using each alkylating agent. The relative value when PEITC was added when the value when PEITC was not added in each alkylating agent was taken as 1 was shown.

<Result>
It was possible to measure anti-alkylating activity not only with alkylating agent MNNG but also with other alkylating agents MNU, ENU and DEN.

  <Detection by changing the substrate from ONPG to X-gal (Wako) and visualizing anti-alkyl activity>

Strain E. coli; YN113 W3110 trp ^R lacZ ValR azi TrpE929 (am) trpA9761 (am) :: pMY3

  (1) Pre-culture 1 Using a large test tube, the cells were cultured in 2.5 ml of LB medium (final concentration 50 μg / ml ampicillin sodium salt) at 37 ° C. for 14 hours.

  (2) Preculture 2 Using a 200 ml Erlenmeyer flask, the above was diluted 1/100 with 30 ml of LB medium (final concentration 50 μg / ml ampicillin sodium salt) and cultured at 37 ° C. for 1 hour 50 minutes.

  (3) The alkylating agent MNNG (final concentration 0.005 μg / ml) was added to (2).

  (4) As a test substance, a sample was prepared by diluting so that the concentration of PEITC was 0.5, 1, 2, 5, 10 μg / ml.

  (5) Sample dispensing. 1 ml of (3) was dispensed to the small examiner of (4) and stirred. A sample of each tube was dispensed into a 2 ml 96-well plate at 250 μl per well (4 or 5 wells were dispensed for each small test tube (concentration)).

  (6) Sampling and sample adjustment. Samples were collected from each hole in the plate at 0.5, 1, 2, and 4 hours after addition of the alkylating agent. A 0.5 ml 96-well plate was prepared, and 150 μl of Z buffer was put in advance. Further, 30 μl of each culture sample was added, one drop of toluene was added, and then the lid was capped and vortexed. At the same time, 100 μl of the sample was collected in a 96-well assay plate, and the absorbance at a wavelength of 600 nm was measured with a plate reader to measure the turbidity of the sample.

  (7) Assay. After completion of all sampling, the sample was centrifuged briefly and the lid was opened. The mixture was incubated at 37 ° C. for 30 minutes to evaporate toluene. 50 μl of assay buffer (final concentration 40 μg / ml X-gal in PBS) was dispensed into a 96-well assay plate per sample. To this was added 50 μl of sample and left at room temperature for 90 minutes. The upper diagram of FIG. 5 shows the antialkylation activity at each concentration 1 hour after the start of the main culture when the substrate of this example was changed from ONPG to X-gal, and the lower diagram shows the substrate from ONPG to X-gal. It showed about the photograph of the test plate when changed to.

<Result>
Even when the substrate was changed from ONPG to X-gal, the anti-alkylation activity could be detected in the same manner. ONPG has better sensitivity, but in this example using the substrate X-gal, it turns blue when the substrate is degraded by β-galactosidase, so it is easy to confirm the activity visually. For example, in the absence of a plate reader, anti-alkylating activity can be detected with the naked eye, which is very useful.

<Effects of metabolically activated analytes on alkylation>
S9Mix sample preparation (for metabolic activation) / ml
G-6-P (oriental yeast) 1.7mg
NADPH (Oriental Yeast) 3.62mg
NADH (Oriental Yeast) 3.05mg
0.2MPO ₄ buffer 2.5ml
1 MKCl 33 μl
1 MMgCl ₂ 8 μl
To the above substance, water is added so that the total amount becomes 0.9 ml, and further 100 μl of S-9 (rat liver homogenate) (Kikkoman) is added.

  (1) Preculture 1 Using a large test tube, the cells were cultured at 37 ° C. for 14 hours in 2.5 ml of LB medium (50 μg / ml ampicillin sodium salt).

  (2) Preculture 2 Using a 200 ml Erlenmeyer flask, the above was diluted 1/100 with 30 ml of LB medium (50 μg / ml ampicillin sodium salt) and cultured at 37 ° C. for 1 hour 50 minutes.

  (3) Preparation of metabolic activation samples. To the cells in (2), S9Mix (final concentration 10%) was added, and further an alkylating agent MNNG (final concentration 0.005 μg / ml) was added (a sample in which S9 was not added to the control was prepared).

  (4) As test substances, samples were prepared by diluting the cysteine (cystiamine (Wako)) concentration to 1, 2, 5, 10, 25 μg / ml.

  (5) Sample dispensing. 1 or 1.5 ml of (3) was dispensed to the small examiner of (4) and stirred. Samples of each tube were dispensed into a 2 ml 96-well plate at 250 μl per well (4 or 5 wells were dispensed for each small test tube (concentration)).

  (6) Sampling and sample adjustment. Two hours after the addition of the alkylating agent, a sample was collected from each hole of the plate. A 0.5 ml 96-well plate was prepared, and 150 μl of Z buffer was put in advance. Further, 30 μl of each culture sample was added, one drop of toluene was added, and then the lid was capped and vortexed. At the same time, 100 μl of the sample was collected in a 96-well assay plate, and the absorbance at a wavelength of 600 nm was measured with a plate reader to measure the turbidity of the sample.

(7) Assay. After completion of all sampling, the sample was centrifuged briefly and the lid was opened. The mixture was incubated at 37 ° C. for 30 minutes to evaporate toluene. 50 μl of assay buffer (final concentration of ONPG in Z buffer: 1 μg / ml) per sample was dispensed into a 96-well assay plate. 30 μl of sample was added to this and left at 28 ° C. for 10 minutes. The reaction was stopped by adding 40 μl of 1M Na ₂ CO ₃ . Absorbance at a wavelength of 414 nm and a wavelength of 550 nm was measured with a plate reader.

(8) Activity was calculated | required by the following calculation.
(Anti-alkylation activity) U (unit) = 1000 × (A ₄₁₄ -1.75 × A ₅₅₀ ) / (t × 0.0075 × A ₆₀₀ )
t; time of incubation at 28 ° C. (minutes)

  (9) Data processing. In addition to the comparison of the number of units, the influence of the test substance in each sample when the control was 1 was detected. In FIG. 6, in order to investigate the effect of metabolic activation enzyme S9, the alkylating agent MNNG and the test substance cystiamine were added with and without S9 added, and the antialkylation activity of cystiamine was shown.

  <Result>

  Cysteamine (cystiamine (Wako)), Epicatechin (epicatechin (Sigma)), Phloroglucinol (phorologlucinol (Sigma)), etc. showed anti-alkylating activity by adding metabolic activity S9.

  <Detection of a large number of samples>

  Below, each test substance and each density | concentration of this test substance were measured for the antialkylation at once.

  (1) Preculture 1 Using a large test tube, the cells were cultured at 37 ° C. for 14 hours in 2.5 ml of LB medium (50 μg / ml ampicillin sodium salt).

  (2) Preculture 2 Using a 200 ml Erlenmeyer flask, the above was diluted 1/100 with 30 ml of LB medium (50 μg / ml ampicillin sodium salt) and cultured at 37 ° C. for 1 hour 50 minutes.

  (3) Dilution of the test substance. Fifteen test substances were prepared. The final concentration of the test substance per hole was adjusted to be 1, 2, 5, 10, 25 μg / ml (in this case, the measurement can be facilitated by changing the concentration horizontally).

  (4) The alkylating agent MNNG (final concentration 0.005 μg / ml) was further added to the cells of (2).

  (5) The sample was dispensed. 250 μl of (4) was dispensed into a 2 ml 96-well plate at 250 μl per well on the plate to which the test substance of (4) was added (250 μl may be dispensed after mixing with the test substance in a small test tube) ).

  (6) Sampling and sample adjustment. Two hours after adding the alkylating agent, a sample was collected from each hole of the plate. Prepare 0.5 ml of 96-well plate and add 150 μl of Z buffer in advance. Further, 30 μl of each culture sample was added, one drop of toluene was added, and then the lid was capped and vortexed. At the same time, 100 μl of the sample was collected in a 96-well assay plate, and the absorbance at a wavelength of 600 nm was measured with a plate reader to measure the turbidity of the sample.

(7) Assay. After completion of all sampling, the sample was centrifuged briefly and the lid was opened. The mixture was incubated at 37 ° C. for 30 minutes to evaporate toluene. 50 μl of assay buffer (final concentration of ONPG in Z buffer: 1 μg / ml) per sample was dispensed into a 96-well assay plate. 30 μl of sample was added to this and left at 28 ° C. for 10 minutes. The reaction was stopped by adding 40 μl of 1M Na ₂ CO ₃ . Absorbance at a wavelength of 414 nm and a wavelength of 550 nm was measured with a plate reader.

(8) Determine the activity by the following calculation (anti-alkylation activity) U (unit) = 1000 × (A ₄₁₄ -1.75 × A ₅₅₀ ) / (t × 0.0075 × A ₆₀₀ )
t; time of incubation at 28 ° C. (minutes)

  (9) Each activity was calculated when the activity of the sample without the control test substance was set to 1.

  <Result>

  Using 15 types of test substances, various concentrations of anti-alkylation activity could be measured. With such a configuration, the detection method of the present invention can achieve high throughput.

  FIG. 1 is a diagram showing the antialkylation activity of the alkylating agent MNNG and the test substance PEITC for 1 hour.   FIG. 2 shows the anti-alkylation activity of pre in which the alkylating agent MNNG is added before the addition of the test substance β-carotene and the post in which the alkylating agent MNNG is added after the addition of the test substance. It is.   FIG. 3 shows the anti-alkylation activity over time at various concentrations of the test substance PEITC against naturally occurring alkylation.   FIG. 4 is a diagram showing a comparison of the anti-alkylating activity with the test substance PEITC using each alkylating agent.   The upper diagram of FIG. 5 shows the antialkylation activity at each concentration 1 hour after the start of the main culture when the substrate of this example was changed from ONPG to X-gal, and the lower diagram shows the substrate from ONPG to X-gal. It is the figure shown about the photograph of the test plate at the time of changing to.   FIG. 6 is a diagram showing the anti-alkylating activity of cystiamine by adding an alkylating agent MNNG and a test substance cystiamine in the presence and absence of S9 to examine the effect of metabolic activation enzyme S9. is there.

Claims (17)

Adding an alkylating agent to the host cell;
And a step of adding a test substance to the host cell.   The method for searching for an anti-alkylating substance according to claim 1, wherein the host cell has a fusion gene of an alkylation repair gene and a gene serving as an index.   The method for searching for an anti-alkylating substance according to claim 1 or 2, wherein the host cell is derived from E. coli.   The method for searching for an anti-alkylating substance according to any one of claims 1 to 3, wherein the alkylation repair gene is an E. coli ada gene.   The method for searching for an anti-alkylating substance according to any one of claims 1 to 4, wherein the index gene is Escherichia coli lacZ.   6. The method for searching for an anti-alkylating substance according to claim 1, wherein the host cell DNA is alkylated in the step of adding the alkylating agent.   The method for searching for an anti-alkylating substance according to any one of claims 1 to 6, wherein the oxygen atom at the 6-position of the guanine base on the DNA is alkylated in the step of adding the alkylating agent.   The alkylating agent is MNNG (1-methyl-3-nitro-1-nitrosoguanidine; 1-Methyl-3-nitro-1-nitrosoguanidine), MNU (N-nitroso-N-methylurea; N-Nitroso-N- 8. The method according to claim 1, which is methylurea), DEN (N-nitrosodiethylamine; N-nitrosodiethylamine) or ENU (N-nitroso-N-ethylurea; N-nitroso-N-ethylurea). Search method for anti-alkylating substances. In the case of adding the test substance before adding the alkylating agent,
9. The method for searching for an anti-alkylating substance according to claim 1, wherein alkylation of the DNA is suppressed. When adding the test substance after adding the alkylating agent,
9. The method for searching for an anti-alkylating substance according to claim 1, wherein alkylation of the DNA is suppressed.   An anti-alkyl comprising a step of adding a test substance to a host cell that is naturally alkylated, wherein the host cell has a fusion gene of an alkylation repair gene and an indicator gene Search method for chemical substances.   The method for searching for an anti-alkylating substance according to claim 11, wherein the host cell is derived from E. coli.   The method for searching for an anti-alkylating substance according to claim 11 or 12, wherein the alkylation repair gene is an E. coli ada gene.   The method for searching for an anti-alkylating substance according to claim 11, wherein the gene serving as an index is Escherichia coli lacZ.   The method for searching for an anti-alkylating substance according to claim 11, wherein the oxygen atom at the 6-position of the guanine base on the DNA is naturally alkylated.   A method for searching for an anti-alkylating substance comprising a step of adding an alkylating agent to a host cell and a step of adding a test substance to the host cell, or a method for detecting a host cell that is naturally alkylated. A method of searching for an anti-alkylating substance comprising a step of adding a test substance, wherein the host cell is derived from E. coli and has an anti-alkylating substance having a fusion gene of an alkylation repair gene and an indicator gene. An anti-alkylating agent characterized by being searched by a searching method.   Containing captopurine, chitosan, chlorophyllin, L-citrulline, curcumin, fisetin, L-glutathione, phenethyl isothiocyanate, quercetin, ascorbic acid, rutin, cystiamine, epicatechin, β-carotene, chitosan and / or phoroglucinol Anti-alkylating agent characterized.

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