JP2012139169A - Mutagenicity test method using mammal cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a high sensitive evaluation method for mutagenicity of chemical and physical mutagens.SOLUTION: The high sensitive quantitative method for mutagenicity of physical or chemical mutagens includes: collecting mammal cells cultured under a prescribed condition during a period within doubling time or till the cells are divided a prescribed number of times under the presence or absence of physical or chemical mutagens, and extracting a genomic DNA-containing fraction; roughly purifying an amplified product by amplifying DNA by means of random PCR under a prescribed condition using a non-specific primer with the obtained DNA as a template; imaging the result of gel electrophoresis implemented with a prescribed temperature gradient in the direction orthogonally crossing the voltage applying direction; extracting a two-dimensional position of a starting point of separation of two DNAs on an appearing band for normalization and computing the position of species identification dots; computing the amount of divergence of the position of the respective species identification dots under the absence or presence of a specimen; and detecting and determining the quantity of the mutagenicity of the specimen based on the computed amount of divergence.

Description

  The present invention relates to a method for detecting mutagenicity. More specifically, the present invention relates to a highly sensitive method for detecting mutagenicity using mammalian cells.

So far, many chemical substances have been created and used in social life. Under such circumstances, the “Law Concerning the Examination of Chemical Substances and Production Regulations” was enacted in 1973, triggered by environmental pollution problems caused by PCBs. In this way, the newly manufactured or imported chemical substances are examined in advance to determine their harmfulness to humans, and there is a risk that human health may be impaired through the environment. In the case of applicable chemical substances, a mechanism was established to regulate their production, import and use.
Here, it is obliged to conduct a reverse mutation test using bacteria and a chromosomal aberration test (mutagenicity test) using cultured mammalian cells as a screening toxicity test when deciding whether or not it falls under a specified chemical substance. ing.

Among these mutagenicity tests, the Ames test is the test conducted as the guideline for the safety of the most chemical substances or products using them. The test advance histidine synthesis gene was deficient Salmonella ^- are those using, the Salmonella to assess qualitatively and / or quantitatively the mutagenicity of the sample from the frequency causing reverse mutation (His) This is a method for detecting the mutagenicity of a chemical substance based on a change in bacterial phenotype.
Since Salmonella used here lacks the histidine synthesis gene (His ⁻ ), it cannot grow on a medium containing no histidine and does not form a colony. The Salmonella (His ^-) and is brought into contact with the sample and then cultured on agar medium lacking histidine, only bacteria recovered histidine synthesis ability to form colonies (return colony) causing the reverse mutation . That is, the Ames test is a test for detecting the mutagenicity of a chemical substance based on the phenotype of the used bacteria.

In the Ames test, by setting the number of bacteria to be brought into contact with a sample to be constant, the probability that a reverse mutation has occurred can be obtained from the number of reverted colonies, and the strength of mutagenicity of the sample can be evaluated. When a certain chemical substance is metabolized in vivo to become a mutagen, it is incubated with a rat liver extract (S9 mix), a sample containing the metabolite is prepared, and the test is performed in the same manner. The mutagenic strength of can be evaluated.
In this test, not only Salmonella but also Escherichia coli can be used. In either case, the number of colonies of the used strain is more than twice that of the solvent control, and the dose dependency on chemical substances is observed. If it is determined to be positive.
The Ames test is a test developed in 1971. Currently, strains having different histidine synthesis gene variants have been developed and improved so that the strain can be selected according to the mutation to be detected. Specifically, base substitution mutations can be detected by using strains such as TA92, TA100, and TA1535, and frameshift mutations can be detected by using strains such as TA94, TA98, TA1537, and TA1538. it can.

On the other hand, there is a test method for detecting the mutagenicity of a chemical substance not by the phenotype of the bacteria used but by the genetic traits of the bacteria used. This method is called a genome-profiling-based mutation assay (hereinafter sometimes referred to as “GPMA method”).
In this method, Escherichia coli is brought into contact with a mutagen, the genomic DNA is taken out, subjected to electrophoresis with a temperature gradient after amplification, and the mutagenicity of the mutagen is analyzed by analyzing the electrophoretic profile. (Refer to Non-Patent Document 1; hereinafter referred to as “Conventional Example 1”).

  As a technique for confirming mutagenicity to mammals including humans, there is a method described in JP-A-2005-24 (refer to Patent Document 1, hereinafter referred to as “Prior Art 2”). Prior Art 1 produces a recombinant vector comprising a p53 binding sequence that binds to p53, a transcription factor, a minimal promoter that can function in mammalian cells, and a reporter gene linked so that it can be expressed by this promoter, This is a method in which this is introduced into a human-derived host cell to form a transformant, and the mutagenicity is measured using an increase in the expression level of the reporter gene as an indicator.

JP 2005-24

Futakami et al., Journal of Biochemistry 2007 141 (5) 675-686

This is the state of chemical substance regulation. In Europe in December 2006, it was abbreviated as REACH (Registration, Evaluation, Authorization and Restriction of Chemicals). Is also established, and is regulated by the Toxic Substances Control Act (TSCA) in the United States. And it is obligatory to conduct mutagenicity tests when manufacturing and exporting chemical substances.
The Ames test has been used for a long time because it is excellent in terms of reproducibility and small error range of the results obtained compared to other mutagenicity tests. However, Salmonella reversions used in the Ames test include not only those caused by the presence of the sample, but also those that occur naturally. For this reason, in the case of a sample with weak mutagenicity, it is difficult to determine whether the back mutation that has occurred is due to the sample. Moreover, since the detection sensitivity is in the order of ppm, strong mutagens can be detected, but weak mutagens cannot be detected.
In addition, the Ames test can simultaneously detect what type of mutation the sample induces by using a plurality of strains. However, since this method is a method for observing mutagenicity with bacterial phenotypes, it cannot be detected unless the bacteria form colonies, and it takes about one week. Furthermore, even if Salmonella used in the Ames test is highly mutagenic, it may not necessarily show strong mutagenicity in mammalian cells. This is due to the fact that the bacteria and mammalian cells used have different sensitivity to mutagens, and the strength of mutagens between these two cell types is not correlated.
On the other hand, the GPMA method using Escherichia coli described in Conventional Example 1 is superior in that a result can be obtained in a shorter time than the Ames test because it does not require passage of bacteria. However, the detection sensitivity of mutagenic substances is on the order of ppb, but it has the same problem as the Ames test in that the cells used are not mammalian cells.

The prior art 2 is excellent in that mutagenicity can be easily confirmed using human cells, not Salmonella or the like. However, there is a need for operations such as promoter integration and reporter gene ligation, and the concentration at which the compound used (adriamycin) was judged to be positive for mutagenicity was high on the order of μg (ppm), indicating strong mutagenicity. Can be detected, but compounds showing weak mutagenicity have not been detected.
From the above, there is a strong demand for a method that has high sensitivity, can obtain results in a short time, and can evaluate mutagenicity for mammals at low cost.
Furthermore, in recent years, it has been clarified that there is a correlation between the incidence of sunburn and skin cancer, and the development of a method that can evaluate the mutagenicity of physical mutagens as well as chemical mutagens. Is also sought.

A method for quantifying the mutagenic strength of a physical or chemical mutagen using a mammalian cell, each in the presence and absence of the physical or chemical mutagen ( a1) a cell culturing step for culturing the mammalian cells under a certain condition within a doubling time or until a certain number of divisions; and (a2) collecting the cultured mammalian cells in the cell culturing step, DNA fraction extraction step for extracting DNA-containing fractions; (a3) DNA amplification by random PCR using DNA obtained in the DNA fraction extraction step as a template and using non-specific primers under predetermined conditions A random PCR step, and (a4) roughly purifying the amplification product obtained in the random PCR step, and performing gel electrophoresis with a predetermined temperature gradient in a direction orthogonal to the voltage application direction. And electrophoresis step, an extraction step of extracting a two-dimensional position of (a5) the gel electrophoresis step captures a gel after completion of the melting starting point of the double-stranded DNA on emerged band (P _ini), (a6) A normalization process is performed on the extracted two-dimensional position, and a species identification point (species identification point) in a two-dimensional coordinate system having one coordinate axis as a mobility and another coordinate axis perpendicular to the one coordinate axis as a temperature gradient. (b) a first position which is a position of a species identification point of genomic DNA of the mammalian cell cultured in the absence of the test substance, and the test A calculation step of calculating an evaluation amount of deviation from the second position, which is the position of the species identification point of the genomic DNA of the mammalian cell cultured in the presence of a substance, (c) based on the calculated evaluation amount, Detect and determine the mutagenicity of the test substance. And quantification steps of a further comprises physical or chemical sensitive method of quantifying mutagen mutagenicity.

  The random PCR step is preferably repeated at least once under different conditions. In the calculation step for calculating the evaluation amount, a pattern similarity score (hereinafter referred to as “PaSS”) calculated by the following formula (I): ) And ΔPaSS and ΔΔPaSS obtained by the following formulas (II) and (III), it is preferable to calculate the evaluation amount of the deviation.

ΔPaSS = 1-PaSS (II)
ΔΔPaSS = ΔPaSS ^s −ΔPaSS ^R (III)
Here, in formula (I), n represents the number of species identification points, and i represents a specific point among those species identification points. In formula (III), S represents a sample that may contain a mutagen, and R represents a difference ΔPaSS between the starting sample and the target sample treated in the same manner without the mutagen. .

As said physical mutagen, it can use suitably with respect to an ultraviolet-ray. As said chemical mutagen, it can use suitably with respect to a compound or a composition. Examples of the chemical mutagens include ethidium bromide (EtBr), furiframide (AF2), methyl methanesulfonate (MMS), 4 ', 6-diamidino-2-phenylindole (DAPI) and crystal violet (CV). It can be used more suitably for a compound or composition selected from the group consisting of:
The mammalian cell is preferably a cell selected from the group consisting of NIH3T3, CHO, HeLa, HEK293, Neuro2A, COS-1 / 7, and HepG2, and any one selected from the group consisting of NIH3T3, CHO, and HeLa. More preferably, it is a cell.
In the cell culture step, the certain number of times is preferably 3 to 15, and more preferably 3 to 5.

The non-specific primer used in the random PCR step is preferably an 8 to 14-mer oligonucleotide, more preferably a dodecanucleotide, and particularly one having the following sequence (SEQ ID NO: 1). preferable.
dAGAACGCGCCTG
Annealing is preferably performed at 4 ° C. or higher and 40 ° C. or lower as the predetermined condition in the random PCR step, and more preferably performed at 26 to 28 ° C.
The predetermined temperature gradient in the gel electrophoresis step is preferably a minimum of 10 ° C. and a maximum of 80 ° C., more preferably 30 to 70 ° C.
By performing detection under the above conditions, the detection sensitivity of the mutagenicity of the chemical mutagen can be set to the ppb order.

  In the mutagenic high-sensitivity quantification method of the present invention, mutagenicity to animal cells can be detected and quantified with high sensitivity. In addition, the mutagenicity of chemical and physical mutagens can be directly detected and quantified as genetic traits (DNA damage) rather than cell phenotypes.

FIG. 1 is a schematic diagram of random PCR used in the method of the present invention. FIG. 2 is a schematic diagram showing the results of gel electrophoresis (μTGGE) in which a predetermined temperature gradient is applied in the direction orthogonal to the voltage application direction on the migration surface. FIG. 3 is a diagram showing results (A) and (B) obtained by subjecting a random PCR product obtained from Escherichia coli treated with ethidium bromide (EtBr, mutagen) to μTGGE and results obtained by normalizing them. 3A shows the case where the 0th generation is used, and FIG. 3B shows the case where the 5th generation is used. In the figure, the vertical axis (μ) represents mobility, and the horizontal axis (θ) represents temperature. ΔPaSS will be described later. FIG. 4 is a schematic diagram showing a case of analyzing a shift (after normalization processing) of a movement position of a DNA fragment caused by a genomic mutation caused by a mutagen. In (A), the spiddos of P _i are points after gel electrophoresis when cultured under mutagenic absence DNA amplification products, also, Pi 'is the corresponding DNA amplification when cultured under mutagenic presence Represents the product spiddos. (B) is a schematic diagram showing the relationship between the resulting mutation and positional deviation. FIG. 5 is a diagram schematically showing the above formula (III).

FIG. 6 is a view showing the results of observing with a microscope the state of NIH 3T3 cells after 5-generation culture when treated with 0.3 to 10 ppm ethidium bromide (EtBr, mutagen). FIG. 6 (A) is a control. FIG. 7 is a graph showing the detection sensitivity of EtBr when NIH 3T3 cells after 5 or 3 generation cultures are used. (A) is after 5 generation culture, (B) is after 3 generation culture. In the figure, * indicates that the cells are almost dead.

FIG. 8 is a photomicrograph when the following test substance is added and cultured using HeLa cells. (A) Negative control, (B) Ethidium bromide (EtBr), (C) Furyl flamide (AF2), (D) 4 ', 6-diamidino-2-phenylindole (DAPI), (E) Crystal violet ( Crystal Violet), and (F) Methyl methanesulfonate (MMS). FIG. 9 is a graph showing the ΔPaSS value of DNA extracted from each cultured cell of FIG.

The present invention is described in further detail below.
The method for quantifying the mutagenicity of a physical or chemical mutagen of the present invention comprises at least the following (a1) to (a6) steps, and the mutation of a physical or chemical mutagen using mammalian cells. It quantifies the originality. Here, in the step (a1) which is a cell culturing step, the mammalian cells are cultured under a certain condition within a doubling time or until a certain number of divisions; a DNA fraction extraction step (a2) In the step, the mammalian cells cultured in the cell culturing step are collected and a genomic DNA-containing fraction is extracted. In step (a3), which is a random PCR step, DNA amplification is performed by random PCR under a predetermined condition using the DNA obtained in the DNA fraction extraction step as a template and using non-specific primers; gel electrophoresis In the step (a4), the amplification product obtained in the random PCR step is roughly purified, and gel electrophoresis is performed with a predetermined temperature gradient perpendicular to the voltage application direction. Next, in step (a5), which is an extraction step for extracting a two-dimensional position, the gel is imaged after completion of the gel electrophoresis step, and the two-dimensional melting start point (P _ini ) of the double-stranded DNA on the appearing band. A position is extracted; a normalization process is performed on the extracted two-dimensional position in step (a6), which is a calculation step of calculating the position of species identification dots, and one coordinate axis is moved to a mobility And the position of the seed identification point in the two-dimensional coordinate system having the temperature gradient on another coordinate axis perpendicular to the one coordinate axis is calculated.

  The method for quantifying the mutagenicity of a physical or chemical mutagen of the present invention further comprises the step (b) which is a step of calculating an evaluation amount of the following deviation and the step (c) which is a quantification step. It may be provided. Here, in the step (b), a first position which is a position of a species identification point of genomic DNA of the mammalian cell cultured in the absence of the test substance, and the culture cultured in the presence of the test substance Calculating an evaluation amount of deviation from the second position, which is the position of the species identification point of the genomic DNA of the mammalian cell; in step (c), based on the calculated evaluation amount, the mutagen of the test substance Detect and quantify sex.

The mammalian cell used in the step (a1) of the present invention is not particularly limited as long as it can be grown in vitro at a predetermined temperature. As mammalian cells, cell lines derived from mice, rats, Chinese hamsters, humans and other mammals can be used, and clinically obtained cells can also be used if they can be cultured. Mammalian cells to be used can be either adherent cells or suspension cells.
Examples of cell lines derived from mammals include NIH 3T3, CHO, HeLa, HEK 293, Neuro 2A, COS-1 / 7, and Hep G2. Among these, NIH 3T3, CHO, HeLa It is preferable to use HEK 293 and Neuro 2A because physical mutagen or chemical mutagen can be detected and quantified with high sensitivity using DNA damage as an index.

  Here, NIH 3T3 is an established cell line isolated from the fetal skin of NIH Swiss mice. CHO is an established cell line isolated and established from Chinese hamster ovary cells. HeLa is a cell line isolated and established from human cancer tissue. HEK 293 is a cell line established by transforming human fetal kidney cells with the E1 gene of adenovirus. Neuro 2A is a cell line isolated and established from mouse neuroblastoma.

These cells are cultured within the doubling time or until a certain number of divisions. Here, “doubling time” refers to the time required for the number of cells to double (doubling time). In addition, “a certain number of times” means the number of times that a cell divides, and does not mean the number of times that a cell has been passaged.
The predetermined number is preferably 3 to 15 times. If it is less than 3 times, the detection sensitivity does not increase so much, and if it exceeds 15 times, no further improvement in detection sensitivity is expected. Since the detection sensitivity of a mutagen becomes high, it is more preferable that it is 3-5 times. The number of cells used in the method of the present invention is determined by the relationship between the area of the well of the plate to be used and the cell size. For example, when NIH 3T3 cells are used, 1 to 3 × 10 ⁴ It is preferable to do.

Examples of the physical mutagen include ultraviolet rays, radiation, electromagnetic waves, and the like, and examples of the chemical mutagen include ethidium bromide, furiframide, methyl methanesulfonate, dioxin, benzaldehyde, 4 ′, 6- Examples thereof include diamidino-2-phenylindole and other compounds and compositions containing them, such as tar and meat burns. Hereinafter, these chemical mutagens are collectively referred to as “test substances”.
When these substances are used as test substances and a mutagenicity test is performed by the method of the present invention, mutagens having a low concentration that cannot be detected by the Ames test can be detected and quantified.
Furthermore, it is weakly mutagenic against Salmonella and Escherichia coli, and as a result it is not determined to be mutagenic, but in fact, it is a chemical mutagen that has mutagenicity for mammalian cells. Can be detected appropriately. In addition, the strength of their mutagenicity can also be quantified, and based on the result, the effect on humans can be simulated.
As a result, it is possible to detect and quantify low-concentration mutagens contained in drinking water such as tap water, distilled water and other water, rivers, well water, mineral water and coffee, and other food and drink with high sensitivity. Become.

In the step (a2), the mammalian cells cultured under certain conditions in the step (a1) are collected, and a genomic DNA-containing fraction is extracted from the cells according to a conventional method. “Constant conditions” here refers to the concentration of the test substance, the composition of the culture medium, the culture temperature, the culture time, and the like. If the concentration of the test substance is, for example, on the order of 1 ppb to 1,000 ppb, even if the mutagenicity of the mutagen at a low concentration that cannot be detected by the Ames test that detects by the phenotype trait can be accurately detected as a genetic trait There is an advantage.
The culture medium can be appropriately selected and prepared according to the mammalian cells to be cultured. For example, Earl MEM (hereinafter sometimes referred to as “MEM”), Dulbecco's Modified MEM (hereinafter also referred to as “D-MEM”), RPMI 1640, and other media, inactivated calf serum ( Hereinafter, antibiotics and other substances such as fetal serum (hereinafter sometimes referred to as “FBS”), penicillin, or streptomycin can be used as appropriate. The culture temperature is preferably 32 to 38 ° C. and about 5% CO ₂ atmosphere in order to appropriately grow the cultured cells.

In the step (a3), amplification is performed by random PCR under predetermined conditions using the DNA obtained in the DNA fraction extraction step as a template and using predetermined primers. The DNA obtained in the DNA fraction extraction step is the total DNA including satellite DNA and organelle DNA obtained at the time of extraction from cells. In the present specification, these DNAs are collectively referred to as “genomic DNA”. .
In this specification, random PCR is performed by setting the annealing temperature to a low temperature of about 40 ° C. and utilizing the fact that primers bind to multiple sites of DNA extracted as described above, including mismatches. PCR that randomly samples a partial sequence of DNA. FIG. 1 schematically shows this random PCR.
In this random PCR, various primers including a commercially available primer can be used and are not particularly limited. Among these, 8- to 14-mer oligonucleotides labeled with Cy3 or other fluorescent compounds are preferable because they can bind nonspecifically to many sites on genomic DNA, and dodecanucleotides are used. More preferably.

As such a labeled dodecanucleotide, for example, pfM12 (SEQ ID NO: 1; dAGAACGCGCCTG) can be preferably used. When these primers are used, various amplifications can be achieved without using multiple types of primers by controlling the annealing temperature and other conditions so as to bind to multiple sites of the DNA to be amplified, including mismatches. The product can be obtained.
Further, the predetermined condition is that annealing at 4 ° C. or more and 40 ° C. or less is preferable because it can bind nonspecifically to a large number of DNA sites to be amplified, and further, 10 ° C. or more and 32 ° C. or less. It is preferable to carry out with. A temperature of about 28 ° C. is most preferred for reasons of standardization of experimental conditions for various organisms.

The amplification product thus obtained is roughly purified according to a conventional method in step (a4), and is subjected to genome profiling by gel electrophoresis. As schematically shown in FIG. 2, in gel electrophoresis performed in the step (a4), a predetermined temperature gradient is given in the direction orthogonal to the voltage application direction on the migration surface. The predetermined temperature gradient is preferably a minimum temperature gradient of 10 ° C. and a maximum temperature gradient of 80 ° C., since it can be accommodated without obtaining the entire heat denaturation process of DNA, and a temperature gradient direction of 30 ° C. to 70 ° C. It is further preferable in terms of improving the resolution.
The amplification product (double-stranded DNA) that has been subjected to gel electrophoresis begins to melt partially into a single-stranded state at a certain temperature in the gel with a temperature gradient. A remarkable change appears in the electrophoretic pattern (see FIG. 2). The point at which melting of this double-stranded DNA begins is called the melting start point (P _ini ).

In step (a5), the gel is imaged after completion of the gel electrophoresis step, and the position of the melting start point of each amplification product is specified and extracted as a two-dimensional position appearing in the imaged electrophoresis result (FIG. 3 (A) and (B)).
The two-dimensional position of the melting start point (P _ini ) is determined for each genome sequence, and it has been experimentally clarified that reproducibility is high under similar conditions. 3 (A ′) and (B ′) show the results of gel electrophoresis when E. coli is used, the same results are obtained when mammalian cells are used. As described above, the two-dimensional position of P _ini of each amplification product is extracted.
In the step (a6), the two-dimensional position of P _ini is extracted as described above, and the normalization process is performed on the two-dimensional position where each amplification product is extracted using the coordinate information of the internal reference sample. Then, the position of species identification dots (hereinafter, sometimes referred to as “spiddos”) in a two-dimensional coordinate system in which one coordinate axis is mobility and another coordinate axis perpendicular to the one coordinate axis is temperature. calculate.
Here, spiddos is specific to the base sequences of the primer and template DNA, and is a species-specific parameter. Here, spiddos represents species or has species-specific properties, and a species means a cell having the same genome.
If a mutation such as base insertion, deletion, G / C to A / T substitution or vice versa has occurred in the DNA obtained as described above, it is compared with DNA without such mutation. In this case, the position of the species identification point changes. For example, when there is a deletion / insertion, the mobility (μ) changes, and when a point mutation occurs from G / C to A / T or vice versa, the change occurs along the temperature coordinate axis (θ). This occurs (see FIGS. 4A and 4B). Thus, a position P _i of the mutation without species identification point, the position of certain identification point mutation is assumed to be P _{i ',} mutations occurring here, be assessed by the Δμ and Δθ It will be possible.

In the normalization, the two-dimensional position of the melting start point of the internal reference sample placed together with the amplification product on each electrophoresis gel is used as an index in order to increase the accuracy of the experiment. As the internal reference sample, a double-stranded DNA having a specific sequence and length, for example, one having the following sequence (SEQ ID NO: 2) can be used.
To determine the spiddos of each sample, two-dimensional plane coordinates of temperature-mobility are corrected based on the two feature points (notable inflection points, etc.) given by the internal reference sample. Get the coordinates of each spiddos (species identification point) under the system. Specifically, coordinate conversion is performed in which the actually measured coordinates of the feature points P _ini, 1 and P _ini, 2 serving as the two internal reference samples correspond to the previously obtained coordinates (theoretical values). This coordinate transformation is performed on the measured coordinates of the characteristic points of each sample to obtain normalized spiddos.
From the obtained value, PaSS is calculated from the following formula (I). Next, using PaSS, ΔPaSS and ΔΔPaSS are obtained according to the following formulas (II) and (III). If spiddos is the same, the PaSS value is 1.

ΔPaSS = 1-PaSS (II)
ΔΔPaSS = ΔPaSS ^S −ΔPaSS ^R (III)
Here, in formula (I), n represents the number of species identification points (spiddos), and i represents a specific point among the species identification points. In the formula (III), S represents the difference (ΔPaSS ^S ) between the sample that may contain the mutagen and the starting sample, and R is a control that was treated in the same way without the mutagen. The difference (ΔPaSS ^R ) from the sample at the start of the sample is shown. The relationship between ΔPaSS ^S and ΔPaSS ^R and ΔΔPaSS is schematically shown in FIG.
As described above, based on the PaSS value obtained using animal cells cultured in the presence and absence of mutagen, the mutagenic strength of the mutagen contained in the sample is determined. To do.

Hereinafter, the case where NIH 3T3 cells are used as animal cells and ethidium bromide is used as a chemical mutagen (test substance) will be described in more detail.
NIH 3T3 cells are cultured in Dulbecco's modified Eagle's medium containing fetal bovine serum (hereinafter sometimes referred to as “FBS”) at a predetermined concentration, for example, 5 to 15%, and dispensed into vials to obtain a liquid. Use one that has been frozen and stored in nitrogen.
Remove the cryopreserved NIH 3T3 cell vial, thaw in a water bath at approximately 36-45 ° C., and transfer to a sterile tube. A predetermined amount, for example, 10 mL of DMEM is added to this sterilized tube, and centrifuged at 1,000 to 1,500 rpm (rotor diameter: about 15 cm, about 170 × g to 380 × g) at room temperature for about 5 minutes.

Remove the centrifugal supernatant by aspiration, add a predetermined amount (for example, 10 mL) of DMEM again to suspend the cells, centrifuge under the same conditions as above, and remove the centrifugal supernatant two or three times. DMSO contained in the culture medium of the cryopreserved cells is excluded. After the final centrifugation, the supernatant is removed and a predetermined amount of medium (for example, 10 mL of 10% FBS-containing DMEM) is added to prepare a NIH 3T3 suspension.
Next, using a hemocytometer, the number of cells is subjected to a viable count under a phase contrast microscope, and it is confirmed that there is a sufficient number of cells (for example, about 10 ⁶ / mL) for culturing.
Next, a predetermined amount of medium (for example, about 5 to 10 mL of 10% FBS-containing DMEM) is dispensed into a 75 cm ² culture flask, and 10 mL of the above cell suspension is added thereto. Incubate for about 48 to about 72 hours at% CO ₂ until the NIH 3T3 cells are confluent.

After confirming that NIH 3T3 cells have become confluent with a microscope, the medium is aspirated and removed, a predetermined solution is added in a predetermined amount to wash the cell surface, and the medium remaining in the flask is removed. For example, it can be washed with 10 mL of phosphate buffered saline (hereinafter referred to as “PBS”). The PBS is removed by suction, a predetermined amount of trypsin treatment solution is added, and trypsin treatment is performed under predetermined conditions. For example, about 9 μL of PBS can be added to an aqueous solution obtained by adding about 90 μL of 2% EDTA (sodium ethylenediaminetetraacetate) to about 900 μL of about 0.2-0.3% trypsin aqueous solution to make a solution for trypsin treatment. . Using such a solution, for example, trypsinization can be performed for several minutes at 37 ° C. in the presence of 5% CO ₂ .
After a few minutes, trypsinization is stopped by adding a predetermined amount of medium. For example, trypsinization may be stopped by adding 500 μL of DMEM (without FBS). It is confirmed whether NIH 3T3 cells are completely separated using a microscope. If they are not separated, pipetting can be performed to completely separate the cells.
Thereafter, centrifugation is performed under the same conditions as described above, and the supernatant containing trypsin and EDTA is removed. A predetermined amount of medium, for example, 10 mL of 10% FBS-containing DMEM is newly added thereto to prepare a cell suspension for passage.

A predetermined amount of medium is dispensed into a new culture flask (for example, a culture flask having a bottom area of 75 cm ² ), and a predetermined amount of the cell suspension is seeded to perform subculture. For example, about 10 mL of 10% FBS-containing DMEM is placed in a culture flask, and about 5 mL (the number of cells is about 1 × 10 ^{6 to} 5 × 10 ⁶ cells / mL) is seeded and subcultured. You can do it. When the subcultured cells become confluent, the cells are treated with a trypsin treatment solution containing trypsin and EDTA as described above to prepare a cell suspension. The NIH 3T3 cells contained in the cell suspension obtained here are designated as the 0th generation.
NIH 3T3 cells are cultured for 3 to 5 generations using an appropriate culture plate such as a 12-well plate. Each well is seeded with 1-3 × 10 ⁴ cells, and about 0.01-10 ppm ethidium bromide is added to the test medium.

Place a negative control group to which ethidium bromide, a mutagen, is not added, on the same plate. The test group is added with varying mutagen concentrations. For example, about 3-100 ng / mL and about 30-300 ng / mL ethidium bromide can be added as final concentrations.
The plate is cultured for a desired period of time, for example, at 37 ° C. and 5% CO ₂ until the desired number of cells in the first to fifth generations reaches the desired number. The culture period can be, for example, one week. The number of cells in each generation is obtained from the following formula, and when cells reach the desired number of generations n, trypsinization is performed as described above to collect the cells.
N = N ₀ × 2 ⁿ

The NIH 3T3 cells collected by trypsinization as described above are then centrifuged under the desired conditions. For example, it can be centrifuged at about 400 to about 600 × g for several minutes at about 2 to about 6 ° C. The centrifugation supernatant is removed, the pellet is washed an appropriate number of times, resuspended in a desired amount of an appropriate solvent, and centrifuged under the same conditions. For example, the pellet can be washed twice, resuspended in 1 mL ice-cold PBS (pH 7.3) and centrifuged.
Finally, the resulting pellet is resuspended in the desired volume of digestion, dispensed into a tube with a lid of about 1.5 mL, capped tightly and incubated with shaking for the desired time. As the digestive fluid used here, for example, 1M NaCl, 1M Tris-HCl, 0.5% EDTA, 5% SDS, 1 mg / mL, resuspended in a digestive fluid containing proteinase K prepared at the time of use, and about 12 Incubate for about 18 hours at about 40-60 ° C. with shaking.

Thereafter, an appropriate amount of an equilibrated phenol / chloroform / isoamyl alcohol mixed solution having a desired mixing ratio is added to perform phenol extraction of DNA. For example, a mixed solution of phenol / chloroform / isoamyl alcohol = 25/24/1 (volume ratio) may be prepared, and this mixed solution may be added in an equal volume. During phenol extraction, centrifuge at the desired conditions and transfer the resulting aqueous phase (upper layer) to a new tube. As desired conditions, for example, centrifugation can be performed at room temperature for about 10 to 30 minutes at 8,000 to 12,000 rpm (10,000 to 24,000 × g).
Ammonium acetate and ethanol are added here and centrifuged. For example, about 1 to 3 to about 2/3 volume of 5-10 M ammonium acetate and 1 to 3 volume of 100% ethanol are added and about 10,000 to 20,000 rpm at about 2 to 6 ° C. for 5 to 15 minutes. Centrifugation can be performed at 17,000-67,000 × g). The resulting pellets can be rinsed, for example, about 70% ethanol for a suitable number of times, for example twice, and then air dried.

Finally, the DNA obtained as described above is resuspended in an appropriate buffer (for example, Tris / EDTA buffer, hereinafter referred to as “TE buffer”), DNA concentration is measured using an absorptiometer. A DNA solution is adjusted to a predetermined DNA concentration, and an aliquot of this DNA solution is used for random PCR.
As a primer for random PCR, a fluorescently labeled primer having a length of 10 to 20 bases can be used. For example, when 12-base-long Cy3-labeled pfM12 (SEQ ID NO: 1; 5′-AGAACGCGCCTG-3 ′) is used, various amplification products can be obtained.
The random PCR reaction solution contains a desired concentration of dNTPs (N = A, T, G, C), magnesium chloride, and Taq polymerase buffer. Specifically, it contains 4 mM dNTPs, 25 mM MgCl ₂ , and a 10-fold concentration buffer for Taq polymerase, and is adjusted to a desired amount, for example, 44.8 μL with distilled water.

In order to minimize the influence of contamination due to contamination with unspecified DNA, this reaction solution is irradiated with ultraviolet rays for about 10 to 30 minutes. Thereafter, a predetermined concentration of template DNA, Cy3-labeled pfM12 and Taq polymerase (manufactured by Biotech International, USA) are mixed in the reaction solution. Next, random PCR is performed using an apparatus such as T Gradient Thermocycler (manufactured by Biometra, UK), PTC-200 (manufactured by MJ-research, USA), Thermal cycler (manufactured by Takara Bio Inc.), or the like. The PCR reaction can be performed at a desired cycle number, for example, 20 to 40 cycles, for example, denaturation at 94 ° C. for 30 seconds, annealing at 28 ° C. for 2 minutes, and elongation at 47 ° C. for 2 minutes.
When the DNA concentration was insufficient, the PCR product obtained was re-amplified when necessary. The PCR conditions were the same as the above random PCR except for the following points. The template DNA may be used by taking 1 μL to 50 μL of the first random PCR product, the annealing temperature may be 60 ° C., and the number of PCR cycles may be 15 cycles. By this re-amplification, the number of DNA bands necessary for setting the species identification point described later and calculating the PaSS value appear in the electrophoretic image of the random PCR product.

The desired DNA can be used as the internal reference sample. For example, the 1341-1540 base site of the fd phage gene VIII (template DNA, hereinafter referred to as “fd-DNA”, SEQ ID NO: 2). Can be amplified and synthesized by standard PCR methods.
For this amplification, for example, MA1 (SEQ ID NO: 3; 5′-TGCTACGTCTCTTCCGATGCTGTCTTTCGCT-3 ′, 5 ′ end is Cy3 labeled, 31 bases in length) is used as a forward primer, and MA2 (SEQ ID NO: 4 is used as a reverse primer). 5′-TTGAATTCTATCGGTTTATCA-3 ′, 21 bases long) can be used.
Prepare a PCR reaction solution having the same composition as above (4 mM dNTPs (N = A, T, G, C), 25 mM MgCl ₂ and 10-fold concentration buffer for Taq polymerase), add distilled water, For example, it can be about 42 μL. UV irradiation is performed in the same manner as in random PCR, and 2 μL of fd-DNA at a predetermined concentration, 10 pmol / mL MA1, 10 pmol / mL MA2, and 1 unit of Taq polymerase are mixed in the reaction mixture and amplified as described above. By doing so, a PCR product of about 200 base pairs can be obtained from the template DNA.

The resulting random PCR product is applied to μTGGE, where conventional temperature gradient gel electrophoresis is minimized to use a 2.5 cm × 2.5 cm × 0.1 cm size gel. By setting the gel to this size, a small amount of sample can be used, and components in the sample can be separated with high accuracy in a short time.
A solution containing urea with a final concentration of 8M, a 40% acrylamide solution with a final concentration of 6% (w / v), and a final concentration of 1 × TBE buffer was prepared. When urea was completely dissolved, tetramethylenediamine (hereinafter, “ TEMED) and 20% ammonium persulfate (hereinafter referred to as “APS”) in the same amount as TEMED, and gently shake. The gel solution is filled in a micro TGGE electrophoresis plate in close contact with the mold to form a well. The number of wells may be 1 (single well) or a plurality of wells.

Also, prepare a 40% acrylamide solution. A desired amount of random PCR product, DNA to be an internal reference sample, and 10% methylene blue dye are mixed in a centrifuge tube of an appropriate size to prepare a sample. The electrophoresis plate filled with the above 6% acrylamide gel is attached to the cassette for μTGGE, and the sample is injected into the well.
Next, a predetermined amount of 1 × TBE (Tris buffer containing EDTA and boric acid) is poured onto the negative electrode and the positive electrode, the μTGGE cassette is placed on the stage of the μTGGE apparatus, and a desired temperature gradient (for example, about 10 minutes at 100 V) Electrophoresis is performed while maintaining a minimum temperature of 15 ° C and a maximum temperature of 55 ° C. After the electrophoresis is completed, the electrophoresis plate is taken out and imaged using a filter for specifying a DNA band, for example, a fluorescence imaging device with an excitation wavelength of 550 nm and a fluorescence wavelength of 565 nm.

Next, a predetermined image analysis is performed on the DNA band pattern imaged as described above, and a set of eight or more feature points is usually assigned to each genome profile shown on the computer. The reason why the number of points to be assigned is 8 or more is that the number of points obtained on average is 8 or more and that the statistical reliability is higher.
Here, an initial melting point is defined as a feature point representing a position vector (see FIG. 1) represented by temperature and mobility. Normalization (normalization) is performed on the initial melting point, and it is converted into species identification points (spiddos) that are parameters specific to the species. To minimize the experimental error that can occur while performing μTGGE, use an internal reference sample with a known sequence as described above, and use the internal reference sample spiddos to initially melt the resulting sample. Normalize the position of the point. The above-described PaSS can be obtained by performing computer processing using a predetermined program and comparing the corresponding control and spiddos of each sample. The amount of PaSS representing the similarity between two genomes is determined using spiddos and can be defined as:

ΔPaSS = 1-PaSS (II)
ΔΔPaSS = ΔPaSS ^S −ΔPaSS ^R (III)
Here, in the formula (I), n represents a species identification point (spiddos), that is, the number of feature points, and i represents a specific point among the species identification points. In formula (III), S is ΔPaSS, which is the difference between the sample that may contain the mutagen and the starting sample, and R is the subject that has been treated in the same way without mutagen. Each ΔPaSS, which is the difference from the sample at the start of the sample, is represented. Find PaSS using the program described in Visual Basic 6. ΔΔPaSS is determined by subtracting the control ΔPaSS ^R from the ΔPaSS ^S for the sample. SR (see FIG. 5).
The PaSS, ΔPaSS and ΔΔPaSS determined as described above can be used to evaluate the strength of chemical mutagens in mammalian cells.

Repeat this process for each species identification point. By performing these operations, for example, there is a relationship between the number of culture generations and detection sensitivity when using NIH 3T3 cells cultured in the desired generation in the presence of the desired concentration of ethidium bromide. It is possible to observe the change in the detection sensitivity itself. Moreover, by confirming such a relationship experimentally, it becomes possible to adjust the number of generations of cultured cells according to the strength of the mutagen, and to perform detection and quantification with high sensitivity.
Hereinafter, the present invention will be described in more detail using examples. However, the present invention is not limited to these examples.

(1) Culture of NIH 3T3 cells The mouse fibroblast cell line NIH 3T3, which is a mammalian cell, was sold from another laboratory in Saitama University. Cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS, manufactured by Invitrogen-Gibco (currently Life Technologies Japan)), dispensed into vials and stored frozen in liquid nitrogen did.
As a mutagen, ethidium bromide [3,8-diamino-5-ethyl-phenylphenanthridium bromide, CAS 1239-45-8] (manufactured by Sigma-Aldrich Japan Co., Ltd.) was used.
Thaw frozen NIH 3T3 cell vials in a 40 ° C water bath, transfer to a sterile tube, add 10 mL of DMEM, and add 1,200 rpm (rotor diameter 15 cm, 240 xg) 5 at room temperature. Centrifuge for minutes.
The operation of removing the supernatant, adding 10 mL of DMEM again to suspend the cells, and centrifuging in the same manner was repeated twice. The centrifugation supernatant was removed, and 10 mL of 10% FBS-containing DMEM was added to prepare a NIH 3T3 suspension.

Using a hemocytometer, the number of cells was viable counted with a phase contrast microscope, and it was confirmed that there was a sufficient number of cells.
In a 75 cm ² culture flask (manufactured by Nippon Becton Dickinson Co., Ltd.) into which about 5 to 10 mL of medium (10% FBS-containing DMEM) was dispensed, 5-10 mL of the above cell suspension was added, 37 ° C., The cells were cultured for 48 to 72 hours under conditions of 5% CO ₂ until the NIH 3T3 cells became confluent.
After confirming that NIH 3T3 cells became confluent, remove the medium by aspiration, add 10 mL of PBS to wash the cell surface adhering to the bottom of the culture flask, and remove the medium remaining in the flask. Rinse. Remove the PBS in the flask by aspiration, add about 900 μL of 0.25% trypsin aqueous solution, about 90 μL of 2% EDTA aqueous solution and 8.9 mL of PBS (all (manufactured by Life Technologies Japan)), Trypsinization was performed for 5 minutes under the condition of% CO ₂ .

After 5 minutes, 500 μL of medium (DMEM) was added to stop the trypsin reaction. Whether or not NIH 3T3 cells were completely separated was confirmed with a microscope. If they were not separated, the cell suspension was repeatedly pipetted to completely separate the cells. Centrifugation was performed under the same conditions as above, the supernatant containing trypsin and EDTA was removed, and 10 mL of 10% FBS-containing DMEM was newly added to obtain a cell suspension.
Subculture was performed by dispensing 10 mL of 10% FBS-containing DMEM in a 75 cm ² culture flask and inoculating 5 mL of the cell suspension. Confluent cells were trypsinized in the same manner as described above to prepare a cell suspension, and NIH 3T3 cells contained in this suspension were designated as the 0th generation.

NIH 3T3 cells were cultured for 3 to 5 generations using 12-well plates (Nihon Becton Dickinson Co., Ltd.). Each well was seeded with 2 × 10 ⁴ cells.
1 μg / mL ethidium bromide was added to the test medium. Of the 12 wells, the first row (n = 4) contained a medium without addition of ethidium bromide and served as a negative control group. In the second and third rows (n = 4), ethidium bromide was added to give different concentrations (0.3 μg / mL and 10 μg / mL, respectively).
The plate was cultured at 37 ° C. and 5% CO ₂ for one week until the desired number of cells of each generation up to the fifth generation was reached. The results of observation with a microscope are shown in FIGS. The number of cells in each generation was obtained from the following formula.
N = N ₀ × 2 ⁿ
When cells reached the desired number of generations, trypsinization was performed as described above, and the cells were collected.

(2) Extraction of DNA from NIH3T3 cells The 3T3 cells treated with trypsin as described above were centrifuged at 500 xg for 5 minutes at 4 ° C. 40 g NaCl, 1 g KCl, 5.75 g Na ₂ PO ₄ .7H ₂ O, 1 g KH ₂ PO ₄ dissolved in water, made up to 500 mL, prepared PBS (pH 7.3), ice Chilled. The centrifugation supernatant was removed and the pellet was washed twice, resuspended in 1 mL ice-cold PBS and centrifuged.
10 mL of 1 M NaCl, 1 mL of 1 M Tris-HCl, 5 mL of 0.5% EDTA, 20 mL of 5% SDS, 0.1 mg / mL of proteinase K prepared at the time of use was added to 100 mL to prepare a digestion solution. Finally, the obtained pellet was resuspended in 300 mL of the digestive juice and dispensed into a 1.5 mL microtube (manufactured by Eppendorf Co., Ltd.). Then it was tightly covered and incubated for 12-18 hours at 50 ° C. with shaking.

Thereafter, a phenol / chloroform / isoamyl alcohol = 25: 24: 1 (volume ratio) mixture was added in an equal volume to perform phenol extraction. During phenol extraction, centrifugation was performed at room temperature for 20 minutes at 10,000 rpm (16,800 × g), and the resulting aqueous phase (upper layer) was transferred to a new tube.
To this, 1/2 volume of 7.5 M ammonium acetate and 2 volumes of 100% ethanol were added, and centrifuged at 15,000 rpm (37,800 × g) at 4 ° C. for 10 minutes. The resulting pellet was rinsed twice with 70% ethanol and allowed to air dry.
Finally, the DNA was resuspended in TE buffer. The concentration of DNA was measured using an ND-1000 spectrometer (manufactured by Nanodrop, USA) and adjusted to the required concentration. An aliquot of DNA solution (approximately 2.5 μL) was used for random PCR, and the rest was stored at −20 ° C.

(3) Random PCR of DNA derived from animal cells
The PCR reaction solution was prepared in a clean bench. PCR was performed using only the fluorescent primer Cy3-labeled pfM12 (SEQ ID NO: 1; 5′-AGAACGCGCCTG-3 ′ 12 base length). To 2.5 μL of 4 mM dNTPs (A, T, G, C), 5 μL of 25 mM magnesium chloride and 5 μL of 10 × Taq polymerase buffer (Biotech International, USA) are added, resulting in 44.8 μL with distilled water. As described above, a PCR reaction solution was prepared.
In order to prevent contamination due to contamination with unspecified DNA, the prepared PCR reaction solution was irradiated with ultraviolet rays for 15 to 20 minutes. Thereafter, 2.5 μL of a predetermined concentration of template DNA, 2.5 μL of 10 pmol / mL Cy3-labeled pfM12, and 0.2 μL of 1 unit Taq polymerase (manufactured by Biotech International, USA) were mixed with this PCR reaction solution.

Random PCR was performed using either TGradient Thermocycler (manufactured by Biometra, UK), PTC-200 (manufactured by MJ-research, USA) or Thermal cycler (manufactured by Takara Bio Inc.). The reaction was performed 30 cycles, with denaturation at 94 ° C. for 30 seconds, annealing at 28 ° C. for 2 minutes, and extension at 47 ° C. for 2 minutes.
When amplification was insufficient, the obtained random PCR product was re-amplified. The PCR conditions were the same as the above random PCR except for the following points. 1 μL of 50 μL of PCR product obtained by the first random PCR was used as template DNA. Re-amplification was performed with an annealing temperature of 60 ° C. and a PCR cycle number of 15. By this re-amplification, the number of DNA bands necessary for setting the species identification point (spiddos), which will be described later, and for calculating the pass value (PaSS value) have appeared in the electrophoretic image of the random PCR product.

(4) Synthesis of DNA as an internal reference sample
The PCR reaction solution was prepared in a clean bench. Amplification was carried out by standard PCR method from the 1341-1540 base sites of the fd phage VIII gene to obtain an internal reference DNA (SEQ ID NO: 2). Specifically, MA1 (SEQ ID NO: 3; 5'-TGCTACGTCTCTTCCGATGCTGTCTTTCGCT-3 ', 5' end is Cy3 labeled, 31 bases long) as a forward primer, and MA2 (SEQ ID NO: 4; 5'-TTGAATTCTATCGGTTTATCA-3 as a reverse primer) ', 21 bases long) was used for amplification respectively.
A reaction solution was prepared by adding 2.5 μL of 4 mM dNTPs (A, T, G, C), 5 μL of 25 mM magnesium chloride, and 5 μL of 10-fold concentration Taq polymerase buffer, and adjusted to 41.85 μL with distilled water. UV irradiation was performed in the same manner as in random PCR, and then 2 μL of the 134-1540 base site of fd phage gene VIII at a predetermined concentration (about 200 μg / mL), 3 μL of 10 pmol / mL MA1, 3 μL of 10 pmol / mL MA2 and 0.15 μL of 1 unit Taq polymerase (manufactured by Biotech International, USA) were mixed.
The reaction was carried out for 30 cycles with denaturation at 94 ° C. for 30 seconds, annealing at 63 ° C. for 60 seconds, and extension at 72 ° C. for 30 seconds for 30 cycles.

(5) Micro TGGE (temperature gradient electrophoresis)
Each of the random PCR products obtained as described above was applied to μTGGE. In order to prepare a gel (2.5 cm × 2.5 cm × 0.1 cm) for μTGGE, a 10 mL gel solution was prepared by the following procedure. First, in a beaker, add 4.8 g of urea (final concentration 8M), 1.5 mL of 40% acrylamide solution (final concentration 6% (w / v)), 1 mL of 10 × TBE buffer (final concentration 1 ×). It was mixed with distilled water and made into a uniform solution using a magnetic stirrer. When urea was completely dissolved, 5 μL of TEMED and the same amount of 20% APS were added and gently shaken.
The gel solution was filled into microTGGE electrophoresis plates (electroplates) in close contact with the mold. 5 to 6 microelectroplates were prepared from 10 mL of gel solution, and these gels were poured into 5 to 6 electrophoresis plates. In this example, a single well electrophoresis plate was used.

A mixture of acrylamide and bisacrylamide (60: 1) was dissolved in distilled water to prepare a 40% acrylamide solution. Dissolve 27% Tris (2-amino-2-hydroxymethyl-l, 3-propanediol), 13.8g boric acid, 10mL 0.5M EDTA (pH8) in distilled water and make 250mL into 10x TBE buffer. Used this. All solutions were prepared at the time of each gel preparation.
6 μL of a random PCR product, 0.3 μL of DNA serving as an internal reference sample, and 0.3 μL of 10% methylene blue dye were mixed in a 0.5 mL centrifuge tube to prepare a sample. As described above, the electrophoresis plate filled with 6% acrylamide gel was attached to the cassette for micro TGGE, and the sample was injected from the upper part of the gel plane.

Next, 600 μL of 1 × TBE was poured into the negative electrode, and 800 μL of 1 × TBE was poured into the positive electrode. The micro TGGE cassette was placed on the stage of a micro TGGE apparatus (Micro TG; manufactured by Taitec Co., Ltd.), the apparatus was turned on, and the sample was electrophoresed at 100 V for 10 minutes. The temperature gradient was maintained at 15-55 ° C. After completion of electrophoresis, the microelectrophoresis plate was taken out and imaged with a fluorescence imaging device (Molecular imager FX, manufactured by BIO-RAD, USA).
A filter having an excitation wavelength of 550 nm and a fluorescence wavelength of 565 nm was used as a filter for specifying a DNA band.

(6) Computer-aided normalization and processing
In this step, the final step of GPMA, a set of 8 or more feature points was assigned to each genomic profile shown on the computer. The initial melting point, which is a feature point representing a position vector expressed by temperature and mobility, was converted into a value inherent to the DNA molecule in the normalization process.
In order to minimize experimental fluctuations that can occur during μTGGE, DNA was used as an internal reference sample with a known sequence, and the results were normalized. PaSS was calculated | required according to the following formula by performing the computer processing and comparing spiddos of a corresponding control and each sample. The results are shown in FIG.

here,
Represents the temperature and mobility vector of each spiddos. The PaSS value was calculated by creating a program with Visual BASIC on a normal personal computer.

FIG. 7 (A) shows the ΔPaSS value when NIH 3T3 cells were cultured for 3 generations in the presence of 0, 1, 10 ppm ethidium bromide, and 5 in the presence of 0, 0.3, 1.0 ppm ethidium bromide. They are shown in FIG. 7 (B) when they are subjected to generation culture. PaSS values listed in the figure represent the average of three independent experiments.
The ΔPaSS value of the negative control group was around 0.02, whereas it significantly increased in the presence of 10 ppm ethidium bromide (mutagen). However, even when the mutagen concentration was 1 ppm and 10 ppm, no significant difference was observed.
In addition, the ΔPaSS value in the presence of 1 ppm ethidium bromide was slightly higher in the 5th generation when compared with the cells cultured in the 5th and 3rd generations, but there was a significant difference between them. (FIGS. 7A and 7B).
From the above results, it was shown that the mutagenicity of the test substance can be detected with high sensitivity in the culture of animal cells of 3-5 generations.

Example 2 Method for Sensitive Determination of Mutagenicity of Chemical Mutagens Using Human-Derived Cells (1) HeLa Cell Culture
Culture was performed in the same manner as in Example 1 using a medium containing 100 ppb of the sample substance and a medium not containing the sample substance. HeLa cells, which are adherent cells, were cultured for three generations, and the state of the cultured cells was observed with a microscope. The results are shown in FIGS.
FIG. 8A is a negative control group cultured without adding the above compound, and FIGS. 8B to 8F are microscopic images of the third generation HeLa cells after each sample was added and cultured. As test substances, ethidium bromide (EtBr, FIG. 8B), furyl flamide (AF2, FIG. 8C), 4 ′, 6-diamidino-2-phenylindole (DAPI, FIG. 8D), crystal violet (Crystal Violet, FIG. 8E), methyl methanesulfonate (MMS, FIG. 8F) was used.

(2) DNA extraction from HeLa cells
DNA extraction from HeLa cells was carried out in the same manner as in Example 1 except for the following description. Cultured cells were collected from each well into an Eppendorf tube with a lid of 1.5 mL, and centrifuged at 12,000 × g for 5 minutes. Thereafter, decantation was performed, and the medium was removed using a pipette. 200 μL of the same digestion solution as used in Example 1 was added thereto and left overnight with stirring at 55 ° C. to digest endogenous proteins and the like.
Next, add 200 μL of equilibrated phenol / chloroform / isoamyl alcohol solution (used in Example 1) in the same volume as the digestion solution, stir, centrifuge for 20 seconds in a tabletop centrifuge, and add an aqueous layer (containing nucleic acid). Only the fraction) part was collected with a pipette. Further, an equal volume of TE buffer was added to the remaining phenol / chloroform / isoamyl alcohol solution, and the same operation was repeated to re-extract DNA and collect it as an aqueous layer.

  The aqueous layers obtained as described above were combined, and ethanol precipitation was performed as follows. 1/10 volume of 3M sodium acetate and 2.5 volumes of 100% ethanol were added to the aqueous layer, stirred well, and allowed to stand at room temperature for 2 to 10 minutes. Then, it centrifuged at 12,000 xg for 10 minutes at 4 degreeC. The supernatant was removed by decantation, and 1 mL of 70% ethanol was added and stirred. Thereafter, the supernatant was discarded and dried using a centrifugal dryer or desiccator.

(3) Random PCR of human cell-derived DNA
Random PCR was performed under the same conditions as in Example 1, and the obtained DNAs were amplified. In addition, when small-scale culture using a 96-well plate or the like is performed, the number of animal cells obtained is small. Therefore, using 1 μL of the first random PCR product as a template, the annealing temperature is 60 ° C, and the number of cycles is Normal PCR was performed 15 times.

(4) GPMA
Internal reference DNA was synthesized in the same manner as in Example 1, and the PCR amplification product was subjected to μTGGE (temperature gradient electrophoresis) under the same conditions as in Example 1. The obtained genome profile was analyzed by computer in the same manner as in Example 1 to quantify their similarity, and the mutagenicity of each chemical substance was evaluated. The results are shown in FIG.
The average of ΔPaSS of the control DNA caused by the natural mutation was about 0.018, whereas the average of ΔPaSS of each sample was 0.025 to 0.040, indicating a higher average value than that of the natural mutation. When a significant difference was examined with p = 0.01, it was revealed that ΔPaSS of each sample was significantly higher than ΔPaSS of the control DNA. This indicates that each test compound acts as a mutagen on HeLa cells at a concentration of 100 ppb.

From the above, it has been clarified that chemical substances that have already been recognized as mutagens by the Ames test show mutagenicity even by GPMA using human-derived cells (HeLa). Furthermore, according to the method of the present invention, it was shown that the presence of a mutagen having a dilute concentration of 100 ppb can be detected and quantified with high sensitivity using human-derived cells.
From Examples 1 and 2 described above, it was shown that there was no contradiction between the results of GPMA using mammalian cells and the results of the Ames test, and there was a correlation between them. Furthermore, it was shown that mutagenicity not only for E. coli but also for cells derived from mammals can be detected and quantified with high sensitivity.

  The present invention is useful in the chemical, food and pharmaceutical fields.

pfM12
MA1
MA2

Claims (11)

A method for quantifying the mutagenic strength of a physical or chemical mutagen using a mammalian cell, each in the presence and absence of the physical or chemical mutagen,
(A1) a cell culturing step of culturing the mammalian cell under a certain condition within a doubling time or until dividing a certain number of times;
(A2) a DNA fraction extraction step of collecting the cultured mammalian cells in the cell culture step and extracting a genomic DNA-containing fraction;
(A3) a random PCR step in which DNA obtained by the DNA fraction extraction step is used as a template, and DNA amplification is performed by random PCR under a predetermined condition using a non-specific primer;
(A4) A gel electrophoresis step of roughly purifying the amplification product obtained in the random PCR step and performing gel electrophoresis with a predetermined temperature gradient in a direction perpendicular to the voltage application direction;
(A5) An extraction step of imaging the gel after completion of the gel electrophoresis step and extracting the two-dimensional position of the melting start point (P _ini ) of the double-stranded DNA on the appeared band;
(A6) Normalization processing is performed on the extracted two-dimensional position so that one coordinate axis is mobility, and a species identification point in a two-dimensional coordinate system having another coordinate axis perpendicular to the one coordinate axis as a temperature gradient A calculation step of calculating the position of (species identification dots),
(B) a first position which is a position of a species identification point of genomic DNA of the mammalian cell cultured in the absence of the test substance, and a genomic DNA of the mammalian cell cultured in the presence of the test substance A calculation step of calculating an evaluation amount of deviation from the second position which is the position of the seed identification point;
(C) based on the calculated evaluation amount, a quantification step of detecting and quantifying the mutagenicity of the test substance;
A highly sensitive method for quantitatively determining the mutagenicity of a physical or chemical mutagen.   The method according to claim 1, wherein the random PCR step is further repeated at least once under different conditions. In the calculation step of calculating the evaluation amount, the deviation evaluation amount is calculated from the pattern similarity score (PaSS) obtained by the following formula (I) and ΔPaSS and ΔΔPaSS obtained by the following formulas (II) and (III). The method for highly sensitive determination of mutagenicity of a physical or chemical mutagen according to claim 1 or 2, wherein the method is calculated.
ΔPaSS = 1-PaSS (II)
ΔΔPaSS = ΔPaSS ^s −ΔPaSS ^R (III)
Here, in formula (I), n represents the number of species identification points, and i represents a specific point among those species identification points. In formula (III), S represents a sample that may contain a mutagen, and R represents a difference ΔPaSS between the starting sample and the target sample treated in the same manner without the mutagen. .   The physical or chemical mutagen mutagen according to any one of claims 1 to 3, wherein the physical mutagen is ultraviolet light, and the chemical mutagen is a compound or a composition. A highly sensitive quantitative method for sex.   The physics according to any one of claims 1 to 4, wherein the mammalian cell is a cell selected from the group consisting of NIH3T3, CHO, HeLa, HEK293, Neuro2A, COS-1 / 7 and HepG2. Sensitive quantification method of the mutagenicity of chemical or chemical mutagens.   The method according to claim 5, wherein the predetermined number of times is 3 to 15 in the cell culture step.   The mutagenicity of the physical or chemical mutagen according to any one of claims 1 to 6, wherein the non-specific primer used in the random PCR step is an 8 to 14-mer oligonucleotide. Highly sensitive quantitative method.   The method according to claim 7, wherein the predetermined primer is a dodecanucleotide. The method according to claim 8, wherein the predetermined primer has the following sequence (SEQ ID NO: 1).
dAGAACGCGCCTG   The highly sensitive quantification of mutagenicity of a physical or chemical mutagen according to claim 1 or 2, characterized in that the predetermined condition in the random PCR step is annealing at 4 ° C or higher and 40 ° C or lower. Method.   The high sensitivity of the mutagenicity of a physical or chemical mutagen according to claim 1 or 2, wherein the predetermined temperature gradient in the gel electrophoresis step is at least 10 ° C and at most 80 ° C. Quantitation method.