JPS63287490A - Method for testing mutagen - Google Patents
Method for testing mutagenInfo
- Publication number
- JPS63287490A JPS63287490A JP62122561A JP12256187A JPS63287490A JP S63287490 A JPS63287490 A JP S63287490A JP 62122561 A JP62122561 A JP 62122561A JP 12256187 A JP12256187 A JP 12256187A JP S63287490 A JPS63287490 A JP S63287490A
- Authority
- JP
- Japan
- Prior art keywords
- mutagen
- reaction
- added
- test method
- activity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 33
- 239000003471 mutagenic agent Substances 0.000 title claims abstract description 16
- 231100000707 mutagenic chemical Toxicity 0.000 title claims abstract description 16
- 230000003505 mutagenic effect Effects 0.000 title claims abstract description 11
- 238000012360 testing method Methods 0.000 title description 19
- 102000005936 beta-Galactosidase Human genes 0.000 claims abstract description 15
- 108010005774 beta-Galactosidase Proteins 0.000 claims abstract description 15
- 238000006243 chemical reaction Methods 0.000 claims abstract description 15
- 230000000694 effects Effects 0.000 claims abstract description 15
- 102000004190 Enzymes Human genes 0.000 claims abstract description 6
- 108090000790 Enzymes Proteins 0.000 claims abstract description 6
- YHQDZJICGQWFHK-UHFFFAOYSA-N 4-nitroquinoline N-oxide Chemical compound C1=CC=C2C([N+](=O)[O-])=CC=[N+]([O-])C2=C1 YHQDZJICGQWFHK-UHFFFAOYSA-N 0.000 claims abstract description 4
- HJRJRUMKQCMYDL-UHFFFAOYSA-N 1-chloro-2,4,6-trinitrobenzene Chemical compound [O-][N+](=O)C1=CC([N+]([O-])=O)=C(Cl)C([N+]([O-])=O)=C1 HJRJRUMKQCMYDL-UHFFFAOYSA-N 0.000 claims abstract 3
- XFOHWECQTFIEIX-UHFFFAOYSA-N 2-nitrofluorene Chemical compound C1=CC=C2C3=CC=C([N+](=O)[O-])C=C3CC2=C1 XFOHWECQTFIEIX-UHFFFAOYSA-N 0.000 claims abstract 2
- 238000010998 test method Methods 0.000 claims description 11
- 241000894006 Bacteria Species 0.000 claims description 5
- 231100000150 mutagenicity / genotoxicity testing Toxicity 0.000 claims description 3
- 241001138501 Salmonella enterica Species 0.000 claims description 2
- AJEAHBZZHSLIQP-UHFFFAOYSA-N 2-nitrofluoren-9-one Chemical compound C1=CC=C2C(=O)C3=CC([N+](=O)[O-])=CC=C3C2=C1 AJEAHBZZHSLIQP-UHFFFAOYSA-N 0.000 claims 2
- WRMNZCZEMHIOCP-UHFFFAOYSA-N 2-phenylethanol Chemical compound OCCC1=CC=CC=C1 WRMNZCZEMHIOCP-UHFFFAOYSA-N 0.000 claims 2
- 230000003287 optical effect Effects 0.000 claims 2
- JUSWGNJYSBSOFM-UHFFFAOYSA-N 1,3,6,8-tetranitro-9h-carbazole Chemical compound C1=C([N+]([O-])=O)C=C2C3=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C3NC2=C1[N+]([O-])=O JUSWGNJYSBSOFM-UHFFFAOYSA-N 0.000 claims 1
- JOERSAVCLPYNIZ-UHFFFAOYSA-N 2,4,5,7-tetranitrofluoren-9-one Chemical compound O=C1C2=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C2C2=C1C=C([N+](=O)[O-])C=C2[N+]([O-])=O JOERSAVCLPYNIZ-UHFFFAOYSA-N 0.000 claims 1
- VHQGURIJMFPBKS-UHFFFAOYSA-N 2,4,7-trinitrofluoren-9-one Chemical compound [O-][N+](=O)C1=CC([N+]([O-])=O)=C2C3=CC=C([N+](=O)[O-])C=C3C(=O)C2=C1 VHQGURIJMFPBKS-UHFFFAOYSA-N 0.000 claims 1
- IHZCVUBSTYOFSJ-UHFFFAOYSA-N 2,7-dinitro-9h-fluorene Chemical compound [O-][N+](=O)C1=CC=C2C3=CC=C([N+](=O)[O-])C=C3CC2=C1 IHZCVUBSTYOFSJ-UHFFFAOYSA-N 0.000 claims 1
- HDVGAFBXTXDYIB-UHFFFAOYSA-N 2,7-dinitrofluoren-9-one Chemical compound C1=C([N+]([O-])=O)C=C2C(=O)C3=CC([N+](=O)[O-])=CC=C3C2=C1 HDVGAFBXTXDYIB-UHFFFAOYSA-N 0.000 claims 1
- 239000002253 acid Substances 0.000 claims 1
- 239000000469 ethanolic extract Substances 0.000 claims 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 abstract description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 abstract description 4
- 229940088598 enzyme Drugs 0.000 abstract description 4
- 108010015776 Glucose oxidase Proteins 0.000 abstract description 2
- 239000004366 Glucose oxidase Substances 0.000 abstract description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 abstract description 2
- 241000607142 Salmonella Species 0.000 abstract description 2
- 229940116332 glucose oxidase Drugs 0.000 abstract description 2
- 235000019420 glucose oxidase Nutrition 0.000 abstract description 2
- 239000008101 lactose Substances 0.000 abstract description 2
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 abstract description 2
- 238000004451 qualitative analysis Methods 0.000 abstract description 2
- 238000004445 quantitative analysis Methods 0.000 abstract description 2
- 239000012085 test solution Substances 0.000 abstract description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 abstract 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 abstract 1
- 239000012531 culture fluid Substances 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 abstract 1
- 244000005700 microbiome Species 0.000 abstract 1
- 239000011369 resultant mixture Substances 0.000 abstract 1
- 239000000126 substance Substances 0.000 description 24
- 230000001580 bacterial effect Effects 0.000 description 14
- 239000000243 solution Substances 0.000 description 11
- 238000002835 absorbance Methods 0.000 description 10
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 238000011156 evaluation Methods 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 230000005778 DNA damage Effects 0.000 description 5
- 231100000277 DNA damage Toxicity 0.000 description 5
- 230000002255 enzymatic effect Effects 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 238000012031 short term test Methods 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- XKMPRDAFNNEZEM-UHFFFAOYSA-N 8-nitro-1-oxidoquinolin-1-ium Chemical compound C1=C[N+]([O-])=C2C([N+](=O)[O-])=CC=CC2=C1 XKMPRDAFNNEZEM-UHFFFAOYSA-N 0.000 description 3
- 206010007269 Carcinogenicity Diseases 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 238000003915 air pollution Methods 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 231100000260 carcinogenicity Toxicity 0.000 description 3
- 230000007670 carcinogenicity Effects 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 239000000428 dust Substances 0.000 description 3
- 239000003239 environmental mutagen Substances 0.000 description 3
- 238000004020 luminiscence type Methods 0.000 description 3
- 238000000691 measurement method Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- KUWPCJHYPSUOFW-YBXAARCKSA-N 2-nitrophenyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1[N+]([O-])=O KUWPCJHYPSUOFW-YBXAARCKSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000011481 absorbance measurement Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 239000000383 hazardous chemical Substances 0.000 description 2
- 239000002955 immunomodulating agent Substances 0.000 description 2
- 229940121354 immunomodulator Drugs 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- -1 promoters Substances 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000007488 tga-medium Substances 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- IQUPABOKLQSFBK-UHFFFAOYSA-N 2-nitrophenol Chemical compound OC1=CC=CC=C1[N+]([O-])=O IQUPABOKLQSFBK-UHFFFAOYSA-N 0.000 description 1
- WGLQHUKCXBXUDV-UHFFFAOYSA-N 3-aminophthalic acid Chemical compound NC1=CC=CC(C(O)=O)=C1C(O)=O WGLQHUKCXBXUDV-UHFFFAOYSA-N 0.000 description 1
- 208000031404 Chromosome Aberrations Diseases 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- 102000002464 Galactosidases Human genes 0.000 description 1
- 108010093031 Galactosidases Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- WQZGKKKJIJFFOK-DVKNGEFBSA-N alpha-D-glucose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-DVKNGEFBSA-N 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 231100000005 chromosome aberration Toxicity 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229950006191 gluconic acid Drugs 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000001114 myogenic effect Effects 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000002250 progressing effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 231100000378 teratogenic Toxicity 0.000 description 1
- 230000003390 teratogenic effect Effects 0.000 description 1
- 101150115617 umuC gene Proteins 0.000 description 1
- 238000003911 water pollution Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
Ａ 産業上の利用分野
この発明は、環境中の有害物質、特に変異原物質の定性
ないしは定量分析の手法に関する。DETAILED DESCRIPTION OF THE INVENTION A. Field of Industrial Application This invention relates to a method for qualitative or quantitative analysis of harmful substances in the environment, particularly mutagens.
Ｂ 発明の概要
この発明は、特定の変異原性試験方法を採用して変異原
物質と菌とを反応させ、この反応によって生成する酵素
につき、突然変異生成過程で重要な役割を果たすと考え
られているｕｍｕ遺伝子の発現の強さをβ−Ｄ−ガラク
トシダーゼの酵素活性を測定することからなる変異原物
質の分析方法に関するものである。B. Summary of the Invention This invention employs a specific mutagenicity testing method to react a mutagen with bacteria, and the enzyme produced by this reaction is thought to play an important role in the mutagenesis process. The present invention relates to a method for analyzing mutagens, which comprises measuring the intensity of expression of the umu gene and the enzymatic activity of β-D-galactosidase.
Ｃ従来の技術
産業の大幅でかつ急速な発展が行なわれているなかで、
化学工業もこの動向に追随して急成長を行なっているが
、このような情勢に伴って大気汚染、水質汚濁が進行し
、また、催奇形性、筋原性、変異原性の要因が増加する
など、環境は多種多様な化学物質に汚染されている。C.With the significant and rapid development of traditional technology industries,
The chemical industry is following this trend and is rapidly growing, but with this situation, air pollution and water pollution are progressing, and teratogenic, myogenic, and mutagenic factors are increasing. The environment is contaminated with a wide variety of chemicals.
これらの原因を構成する化学物質は、単にこれらを廃棄
・焼却処理を行なう場面に防護対策を実施するのみでは
足りず、大気中での化学反応によっても新たな有害物質
が発生し、これらによる汚染も無視できない現状にあり
、環境保全の観点からも有害物質に対するより確かな対
策が必要とされている。It is not enough to simply implement protective measures when disposing of or incinerating the chemical substances that are responsible for these substances; chemical reactions in the atmosphere also generate new hazardous substances, which can cause pollution. This situation cannot be ignored, and more reliable measures against harmful substances are needed from the perspective of environmental conservation.
環境保全の作業は、原則として、
「−一一一一一−１
調査 −評価 −対策
の閉回路が確実に機能すれば必要な安全対策が採られて
いるということができる。As a general rule, environmental conservation work can be said to have taken the necessary safety measures if the closed circuit of ``11111-1 Investigation - Evaluation - Countermeasures'' functions reliably.
環境保全の作業を効率よく推進し、莞全に達成させるた
めには、これらの諸作業、なかんずく、調査や評価作業
が正確かつ迅速に行なわれることが必要である。In order to efficiently promote environmental conservation work and achieve it in Guanquan, it is necessary that these works, especially surveys and evaluations, be carried out accurately and promptly.
一方、生体影晋の観点から見ると、今後ますますその対
応に重要性を増している環境有害物質としては、変異原
物質、プロモーター、イムノモジュレータ−と見なされ
ているが、これら有害物質を簡易かつ高感度に検出する
手法が開発されていないため、環境保全対策の第１段階
である調査が十分行なえず、正確な評価や対策も十分た
てられない現状にある。On the other hand, from the perspective of biological effects, mutagens, promoters, and immunomodulators are considered to be environmentally hazardous substances that will become increasingly important to deal with in the future. In addition, since a highly sensitive detection method has not been developed, the first stage of environmental conservation measures, such as surveys, cannot be conducted sufficiently, and accurate evaluations and countermeasures cannot be taken.
Ｄ 発明が解決しようとする問題点
このような現状を打破するためには、変異原物質、プロ
モーター、イムノモジュレータ−を高感度かつ簡易迅速
に検出する手法を開発し、環境中のこれらの有害物質の
正確な調査・評価手法を確立することが必要である。D. Problems to be solved by the invention In order to overcome this current situation, it is necessary to develop a highly sensitive, simple and rapid method for detecting mutagens, promoters, and immunomodulators, and to detect these harmful substances in the environment. It is necessary to establish accurate survey and evaluation methods.
ところで、従来行なわれていた方法によれば、以下に述
べるような問題点を有しているものであった。However, the conventional methods have the following problems.
問題点 １
大気浮遊粉塵や大気汚染源からの放出物などには、種々
の発癌関連物質が含まれており、これらのモニタリング
や検出にエイムズ（Ａｍｅｓ）法またはその変法を用い
た復帰突然変異試験が広く使用されている。Problem 1: Airborne dust and emissions from air pollution sources contain various cancer-related substances, and the Ames method or its modified methods should be used in reverse mutation tests to monitor and detect these substances. is widely used.
ところで、国際症研究機関（ＩＡＲＣ）の特別委員会は
、１９８２年にＩＡＲＣモノグラフ１〜２９＠に集録さ
れた化学物質等のヒトに対する発癌性の評価に短期テス
トの結果も考慮に入れることを決定した。By the way, in 1982, a special committee of the International Agency for Research on Diseases (IARC) recommended that results of short-term tests should be taken into consideration when evaluating the carcinogenicity of chemical substances to humans, which were collected in IARC Monographs 1-29@. Decided.
発癌性の予測に関して十分な証拠のある短期テストとは
、
ｒＤＮＡ傷害、突然変異、染色体異常の３種のカテゴリ
ーに属する短期テストのうち、少なくともカテゴリーの
異なる２種以上のテストを用いて、３つ以上の陽性結果
となるか否かを判定するものである。同一カテゴリーに
属するテストでの２つの陽性結果を評価に用いるときは
、異なる生物系を用いた実験での結果でなければならな
い」としている。Short-term tests with sufficient evidence for predicting carcinogenicity are defined as short-term tests that have sufficient evidence to predict carcinogenicity. This is to determine whether or not the above result is positive. "When two positive results from tests in the same category are used in an evaluation, they must be from experiments using different biological systems."
このようなＩＡＲＣの短期テストに対する評価基準を考
慮に入れると、大気浮遊粉塵などに含まれる発癌関連物
質をモニタリングするためには、従来の突然変異試験の
ほかにＤＮＡ傷害や染色体異常についての短期テストも
用いる。ことが望ましい。Taking into account IARC's evaluation criteria for short-term tests, in addition to conventional mutation tests, short-term tests for DNA damage and chromosomal aberrations are necessary to monitor carcinogenic substances contained in airborne dust. Also used. This is desirable.
問題点 ２
細菌、化学物質のＤＮ’Ａ傷害を短時間に検出する方法
としてキラーデッツらは、大腸菌ＰＱ３７株を用いるＳ
ＯＳクロモテスト（ｃｈｒｏｍｏｔｅｓｔ）を報告しく
Ｑｕｉｌｌａｒｄｅｔ、Ｐ、、 Ｈｕｉｓｍａｎ、０．
、 Ｄ’Ａｒ１５．Ｒ。Problem 2 As a method for quickly detecting DNA damage caused by bacteria or chemicals, Kiradetz et al.
To report the OS chromotest, Quillardet, P., Huisman, 0.
, D'Ar15. R.
、ｔｌｏｆｎｕｎｇ、Ｍ、、 Ｐｒｏｃ、Ｎａｔｌ、
Ａｃａｄ、Ｓｃｉ、ＵＳＡ、 ７９．５９７１−５９
７５ （１９８２）あるいはＱｕｉｌｌａｒｄｅｔ、Ｐ
、、 Ｈｏｆｎｕｎｇ、Ｍ、、 Ｍｕｔ、Ｒｅｓ、、１
４７．６５−７８（１９８５） ） 、中村らと小円ら
は、サルモネラ菌ＴＡ１５３５／ｐＳＫ１００２株を用
いるｕｍｕ−テストを報告（中村溝−０小田芙光、沖岩
四部、加藤武司１品川日出夫、日本環境変異原学会、第
１２回大会講演要旨集 Ｐ３９（１９８３）、中村溝−
９小田美光、沖岩四部、中田篤男９品川日出夫、日本環
境変異原学会、第１４回大会講演要旨集Ｐ７１ （１９
８５） 、小円英光、中村清−３沖岩四部、中田篤男１
品川日出夫、環境変異原研究、６．８７−９２（１９８
４）、Ｏｄａ、Ｙ、、 Ｎａｋａｏ＋ｕｒａ、Ｓ、、
Ｏｋｉ、１．、 Ｋａｔｏ、Ｔ、、 Ｓｈｉｎａｇ
ａｗａ、Ｈ，、Ｍｕｔ、１１ｅｓ、、１４７，２１９−
２２９（１９８５） ） Ｌ／ている。, tlofnung, M., , Proc, Natl.
Acad, Sci, USA, 79.5971-59
75 (1982) or Quillardet, P.
, , Hofnung, M., , Mut, Res, , 1
47.65-78 (1985)), Nakamura et al. and Koen et al. reported the umu-test using Salmonella enterica TA1535/pSK1002 strain (Nakamura Mizo-0 Fumitsu Oda, Shibu Okiiwa, Takeshi Kato 1 Hideo Shinagawa, Japan. Environmental Mutagen Society, 12th Conference Abstracts P39 (1983), Mizo Nakamura-
9 Yoshimitsu Oda, Shibu Okiiwa, Atsuo Nakata 9 Hideo Shinagawa, Japanese Society of Environmental Mutagen, 14th Conference Abstracts P71 (19
85), Hidemitsu Koumaru, Kiyoshi Nakamura - 3, Shibu Okiiwa, Atsuo Nakata 1
Hideo Shinagawa, Environmental Mutagen Research, 6.87-92 (198
4), Oda, Y., Nakao+ura, S.
Oki, 1. , Kato, T., Shinag
awa, H,, Mut, 11es,, 147,219-
229 (1985)) L/Iteru.
これらの方法は、共に化学物質のＤＮＡ傷害に伴って生
じるＳＯ５反応を、β−Ｄ−ガラクトシダーゼの活性か
ら求める方法であるが、ＳＯＳクロモテストでは、ＳＯ
Ｓ反応のうち５ｆｉＡまたは５ｕｌＡ遺伝子の発現をβ
−Ｄ−ガラクトシダーゼ活性から求める方法であり、ｕ
ｍｕ−テストではｕｍｕｃ遺伝子の発現をβ−Ｄ−ガラ
クトシダーゼ活性から求める方法である。Both of these methods calculate the SO5 reaction that occurs due to DNA damage caused by chemicals from the activity of β-D-galactosidase, but SOS Chromotest
In the S reaction, the expression of 5fiA or 5ulA gene is β
-D-galactosidase activity, and u
The mu-test is a method for determining the expression of the umuc gene from β-D-galactosidase activity.
そして、 ■測定方法が簡便で測定時間が数時間と短い。and, ■Measurement method is simple and measurement time is short, just a few hours.
■エイムダ法などと異なって１種類の菌株で、種々の癌
・変異原物質を検出し得る。■Unlike the Aimda method, it is possible to detect various cancers and mutagens using one type of bacterial strain.
■検出感度も比較的高い。■Detection sensitivity is also relatively high.
■エイムダ法の結果と比較的良好な相関性を有している
。■It has a relatively good correlation with the results of the Aimda method.
などの特徴を有するものである。It has the following characteristics.
特に、ｕｍｕ−テストは、ＳＯ３反応に関与する遺伝子
群のうち、突然変異生成過程で重要な役割を果たすと考
えられているｕｍｕＣ遺伝子の発現の強さを計測する方
法であるから、発癌関連物質の検出やモニタリングに有
効な方法であると考えられる。In particular, the umu-test is a method to measure the strength of expression of the umuC gene, which is thought to play an important role in the mutation generation process among the genes involved in the SO3 reaction, This is considered to be an effective method for detection and monitoring.
このｕｍｕ−テストは、第４図に示しているように、先
ず、−夜培養した菌液をＴＧＡ培地（バタトトリブトン
、塩化ナトリウム、グルコース、アンピシリンで調整し
たもの）で５０倍に希釈したのち、３７℃で０Ｄ６００
が０．２５〜０．３０に達するまで培養し、この菌液ｚ
、９ｍ１（代謝活性化を必要とする場合は、菌液２．４
ｍｌに５９ｍ１ｘ（エイムズらの方法に従って調整した
もの）０．５ｍｌを加えた液）に被験化学物質０．１ｍ
ｌを加えて３７℃で２時間振盪して培養する。In this umu test, as shown in Figure 4, first, a bacterial solution cultured overnight was diluted 50 times with TGA medium (adjusted with batatotributone, sodium chloride, glucose, and ampicillin), and then 37 0D600 at °C
Culture until z reaches 0.25 to 0.30, and this bacterial solution z
, 9ml (if metabolic activation is required, use 2.4ml of bacterial liquid)
ml and 0.5 ml of 59 ml (prepared according to the method of Ames et al.) to which 0.1 ml of the test chemical was added.
1 and cultured at 37°C for 2 hours with shaking.
このようにして得た菌液のうち、その０．２ｍｌはβ−
Ｄ−ガラクトシダーゼ活性に使用し、残りの２．８ｍｌ
を菌液の濁度（ＯＤ ６００ ）の測定に使用するので
ある。Of the bacterial solution thus obtained, 0.2 ml was β-
Use the remaining 2.8 ml for D-galactosidase activity.
is used to measure the turbidity (OD 600 ) of the bacterial solution.
すなわち、具体的には、被験化学物質と共に振盪培養し
た菌液の一定量（０，２ｍ１）をＺ−緩衝液（１，８ｍ
１）に加え、細胞膜破壊剤（トルエン）を少量加えたの
ち、激しく攪拌振！１（１０秒間）して、菌の細胞膜を
破壊して、β−Ｄ−ガラクトシダーゼを溶出させ、これ
にβ−Ｄ−ガラクトシダーゼの基質であるＯ−ニトロフ
ェニル−β−Ｄ−ガラクトピラノシド（ＯＮＰＧ）を０
゜２ｍｌ加えて３７℃で約２０分間反応させ、１モル／
ｌの炭酸ナトリウム１ｍｌを加えて反応を停止し、分光
光度計により４２０ｎｍと５５０ｎｍの吸光度（ＯＤ４
２０，０Ｄ５５０）をそれぞれ測定し、次式により酵素
活性を算出するものである。That is, specifically, a certain amount (0.2 ml) of a bacterial solution cultured by shaking with a test chemical substance was added to a Z-buffer solution (1.8 ml).
In addition to 1), add a small amount of cell membrane disrupting agent (toluene), then stir and shake vigorously! 1 (for 10 seconds) to disrupt the bacterial cell membrane and elute β-D-galactosidase, which is then treated with O-nitrophenyl-β-D-galactopyranoside (β-D-galactosidase substrate). ONPG) to 0
゜2ml was added and reacted at 37℃ for about 20 minutes, and 1 mol/
The reaction was stopped by adding 1 ml of sodium carbonate, and the absorbance at 420 nm and 550 nm (OD4
20, 0D550), respectively, and the enzyme activity is calculated using the following formula.
ここで、ｔは反応時間（分）、■は反応液中の菌液量の
比（ｏ、２１０．２＋１．ａ）、０Ｄ６００は被験化学
物質と共に振盪培養して得た菌液の６００ｎｍにおける
吸光度を示す。Here, t is the reaction time (minutes), ■ is the ratio of the amount of bacterial liquid in the reaction solution (o, 210.2 + 1.a), and 0D600 is the absorbance at 600 nm of the bacterial liquid obtained by shaking culture with the test chemical. shows.
しかしながら、この方法は、開発されてから日が浅いた
め、測定方法の細部に亘ってまだ十分な検討がなされて
いない。However, since this method was developed recently, the details of the measurement method have not yet been sufficiently studied.
さらにこのｕｍｕ−テストでは、発生したβ−Ｄ−ガラ
クトシダーゼの酵素活性は、化学物質量にも関係し、微
量の化学物質量を検出するためには、β−Ｄ−ガラクト
シダーゼの酵素活性を高感度に計測する必要がある。Furthermore, in this umu-test, the enzymatic activity of β-D-galactosidase generated is also related to the amount of chemical substances, and in order to detect trace amounts of chemical substances, the enzymatic activity of β-D-galactosidase is highly sensitive. It is necessary to measure the
ところが、従来のｕｍｕ−テストの最終検出法は、吸光
度法であるため検出感度が十分であるとは言えない、と
いう根本的なネックを有するものであった。However, the final detection method of the conventional umu-test is an absorbance method, which has a fundamental problem in that the detection sensitivity is not sufficient.
Ｅ 問題点を解決するための手段
従来法によるｉｍｕ−テスト法は、前述のようなもので
あるが、この式から判るように、ミラー（Ｍｉｌｌｅｒ
）の方法は、β−Ｄ−ガラクトシダーゼの０ＮＰＧの作
用により生成したＯ−ニトロフェノールの量を０Ｄ４２
０と０Ｄ５５０の吸光度より求め、これを菌液濃度（０
０６００とＶ）と反応時間で規格化することによりβ−
Ｄ−ガラクトシダーゼの活性を求める方法であり、菌液
中に含まれる化学物質の吸光度は無視している。E. Means for Solving Problems The conventional imu-test method is as described above, but as can be seen from this equation, it is
) method calculates the amount of O-nitrophenol produced by the action of β-D-galactosidase 0NPG by
It is determined from the absorbance of 0D550 and 0D550, and this is calculated as the bacterial liquid concentration (0
β-
This is a method for determining the activity of D-galactosidase, and the absorbance of chemical substances contained in the bacterial solution is ignored.
これに対して、測定精度が高くより利用しやすい測定方
法を確立するために種々検討を行なった結果、この発明
が完成したものである。On the other hand, this invention was completed as a result of various studies to establish a measurement method with high measurement accuracy and ease of use.
今回開発されたこの発明の方法は、第１図にそのフロー
を示したように、（１） Ｚ−緩衝液の成分の１つであ
る２−β−メルカプトエタノールが化学発光を阻害する
という点からＺ−緩衝液の換りに０．１モル／ｌのリン
酸緩衝液（Ｎａ−ＰＢＳ）を用いていること、
（２）菌の細胞膜破壊剤としてクロロホルム（ＣＨＣ１
３）とドデシル硫酸ナトリウム（ＳＯＳ）を用いている
こと、
が異っている。The newly developed method of this invention, as shown in the flowchart in Figure 1, has the following points: (1) 2-β-mercaptoethanol, one of the components of the Z-buffer, inhibits chemiluminescence; (2) 0.1 mol/l phosphate buffer (Na-PBS) was used instead of Z-buffer, (2) chloroform (CHC1
The difference between 3) and 3) is that sodium dodecyl sulfate (SOS) is used.
一般に使用されている菌の細胞膜破壊剤としてのトルエ
ンは、これを蒸発して除去する操作に時間を要するとい
う点があり、あまり好ましいものではない。Toluene, which is commonly used as a cell membrane disrupting agent for bacteria, is not very preferable because it takes time to evaporate and remove it.
また、この発明では、菌液をＴＧＡ培地で５０倍に希釈
した後、０Ｄ６００が０．２５〜０．３０に達するまで
３７℃で培養する時間を節約する工夫がなされている。Moreover, in this invention, after diluting the bacterial solution 50 times with TGA medium, the time for culturing at 37° C. until 0D600 reaches 0.25 to 0.30 is devised to save time.
さらに、β−Ｄ−ガラクトシダーゼ活性を４２０ｎｍと
５５０ｎｍの吸光度を測定して、以下の反応式に示され
るように、化学発光反応により発生する発光量からβ−
Ｄ−ガラクトシダーゼ活性を求めることが従来の方法と
異っている。Furthermore, β-D-galactosidase activity was measured by absorbance at 420 nm and 550 nm, and β-D-galactosidase activity was determined from the amount of luminescence generated by the chemiluminescent reaction, as shown in the reaction formula below.
This method differs from conventional methods in determining D-galactosidase activity.
α−Ｄ−グルコース −一一一一一一
β−Ｄ−グルコース
クルコースオキシダーぞ
β−Ｄ−グルコース＋０２ ＋）ｉ、Ｏ□→Ｄ−グルコ
ン酸十８．０２
アミノフタル酸十Ｎ２ ＋）（、Ｏ＋光大気浮遊粉塵や
大気汚染源からの放出物などには、種々の発癌関連物質
が含まれており、これらのモニタリングや検出にエイム
ダ法またはその変法を利用した復帰突然変異試験が広く
使用されている。α-D-glucose -111111 β-D-glucose glucose oxidizer β-D-glucose +02 +) i, O → D-gluconic acid 18.02 Aminophthalic acid 1N2 +) ( , O+ light Airborne dust and emissions from air pollution sources contain various cancer-related substances, and reverse mutation tests using the Aimda method or its modified methods are widely used to monitor and detect these substances. has been done.
しかし、化学物質によっては例えばＤＮＡ傷害は陽性で
あっても、突然変異は陰性であるなどの結果が得られて
いるので、ＩＡＲＣ（国際癌研究機関）では、化学物質
の評価にあたって、少なくとも２つ以上の項目で陽性の
場合、短期スクリーニングとしての十分な証拠があると
している。However, for some chemical substances, for example, results have been obtained that are positive for DNA damage but negative for mutations, so IARC (International Agency for Research on Cancer) conducts at least two tests when evaluating chemical substances. If the above items are positive, there is sufficient evidence for short-term screening.
以上を総合して勘案すると、この発明では、化学物質の
ＤＮＡ傷害に伴って生じるＳＯＳ反応に関与する遺伝子
群のうち、突然変異生成過程で重要な役割を果たすと考
えられているｕｍｕ遺伝子の発現の強さをβ−Ｄ−ガラ
クトシダーゼの酵素活性から求めるｕｍｕ試験方法に関
するものということができる。Taking all of the above into account, this invention aims to improve the expression of the umu gene, which is thought to play an important role in the mutation generation process, among the genes involved in the SOS reaction that occurs due to DNA damage caused by chemical substances. It can be said that this relates to the umu test method, which determines the strength of β-D-galactosidase from the enzymatic activity.
−Ｆ 実施例
以下、具体的に実施例を示し、この発明の構成 ”およ
び効果をより詳細に説明する。-F Examples Hereinafter, examples will be specifically shown to explain the structure and effects of the present invention in more detail.
実施例
４−ニトロキノリン゛−Ｎ−オキシド（４ＮＱＯ）恋皇
ユ ゛
第１図に示したふうな各工程につき以下に示した手順で
行なった。Example 4 - Nitroquinoline-N-oxide (4NQO) Koioyu Each step as shown in FIG. 1 was carried out according to the following procedures.
■ サルモネラ菌をＬＢ培地で一夜３７℃で培養する。■ Cultivate Salmonella in LB medium overnight at 37°C.
■ ６００ｎｍにおける吸光度を計測し、０Ｄ６００４
０．１になるようにＬＢ培地で希釈調整する。■Measure the absorbance at 600nm, 0D6004
Adjust the dilution with LB medium to a concentration of 0.1.
■ ■で調整した試験液の中に測定対象物である４ＮＱ
Ｏを０．０５ｍ１添加する。■ 4NQ, which is the object to be measured, is in the test solution prepared in ■.
Add 0.05ml of O.
■ ３７℃で２時間培養する。■ Incubate at 37°C for 2 hours.
■ ■の培養液（約３ｍ１）を化学発光測定用と吸光度
測定用に１ｍｌづつバイヤルビンに分取する。② Transfer 1 ml of the culture solution (approximately 3 ml) into vials for chemiluminescence measurement and absorbance measurement.
■ ■のそれぞれのバイヤルビンに０．１モル／ＩＮａ
−ＰＢＳを１ｍｌ、ＣＨＣｌ５０．１ｍｌ、５ＤＳＯ，
１ｍｌを加え、振盪攪拌した後、５分間、静置する。■ 0.1 mol/INa in each vial of ■
-1 ml of PBS, 50.1 ml of CHCl, 5DSO,
Add 1 ml, shake and stir, and then leave to stand for 5 minutes.
■ 吸光度測定用バイヤルビンには、０ＮＰＧ０．２ｍ
ｌを加え、３７℃で１０分間培養する。■ The vial for absorbance measurement contains 0NPG 0.2m.
1 and incubate at 37°C for 10 minutes.
培養後、反応停止液として１モル／ｌのＮａ２Ｃｏ、１
ｍｌを加え、４２０ｎｍにおいて吸光度を計測する。After culturing, 1 mol/l Na2Co, 1
ml and measure the absorbance at 420 nm.
■°一方、化学発光測定用バイヤルビンには、０．１モ
ル／ｌのＮａ−ＰＢＳ２．２５ｍ１．Ｏ。■°Meanwhile, 2.25 ml of 0.1 mol/l Na-PBS was placed in the vial for chemiluminescence measurement. O.
１モル／ｌのＮａＮ、０．５ｍｌ、０．２５モル／１の
ラクトース１ ｍ ｌ 、 ６ ｕ ／ ｍ ｌグルコ
ースオキシダーゼ１ｍｌを加え、３７℃で２時間酵素反
応を行なう。Add 1 mol/l NaN, 0.5 ml, 0.25 mol/l lactose, 1 ml, and 6 u/ml glucose oxidase, and perform an enzymatic reaction at 37°C for 2 hours.
この反応液０．２ｍｌを化学発光測定装置（明電舎製、
ルミノメータｕＰＤ−８０００）専用バイヤルに分取し
、２Ｘ１０−’モル／ｌのルミノール０．５ｍｌと６Ｘ
１０−’モル／ｌのに３Ｆｅ（ＣＮ）ａｏ、５ｍｌを自
動分注操作にして１秒から３０秒間の発光量を計測する
。0.2 ml of this reaction solution was added to a chemiluminescence measuring device (Meidensha Co., Ltd.,
Luminometer uPD-8000)) Transfer to a special vial, add 0.5 ml of 2X10-'mol/l luminol and 6X
5 ml of 10-' mol/l of 3Fe(CN)ao is automatically dispensed and the amount of luminescence is measured from 1 second to 30 seconds.
このようにして測定を行ない、得られた結果を第２図お
よび第３図に示した。Measurements were carried out in this manner, and the results obtained are shown in FIGS. 2 and 3.
第２図は、４ＮＱＯの濃度に対する発光量の挙動を示し
たものであり、また、第３図は４ＮＱＯ濃度に対する吸
光度の挙動を示したものである。FIG. 2 shows the behavior of the amount of luminescence with respect to the concentration of 4NQO, and FIG. 3 shows the behavior of the absorbance with respect to the concentration of 4NQO.
Ｇ 発明の効果
従来行なわれていたｕｍｕ試験方法における最終検出法
は、４２０ｎｍと５５０ｎｍの吸光度を測定し、菌液濃
度と反応時間で規格化してβ−Ｄ−ガラクトシダーゼの
酵素活性を求める方法であったが、本発明は、最終検出
法として化学発光反応により発生する発光量からβ−Ｄ
−ガラクトシダーゼの酵素活性を求めるようにしたので
、従来法に比較して１０〜ｉ ｏｏｏ倍程度の高感度化
を達成することができた。G. Effects of the Invention The final detection method in the conventional umu test method was to measure the absorbance at 420 nm and 550 nm, normalize it by the bacterial solution concentration and reaction time, and determine the enzyme activity of β-D-galactosidase. However, in the present invention, as a final detection method, β-D
- Since the enzymatic activity of galactosidase was determined, it was possible to achieve an increase in sensitivity of about 10 to iooo times compared to the conventional method.
第１図はこの発明によって完成した変異原物質試験方法
の工程図であり、第２および３図は試験結果を示したグ
ラフ、第４図は従来法による変異原物質試験方法の工程
図である。Figure 1 is a process diagram of the mutagen testing method completed by this invention, Figures 2 and 3 are graphs showing the test results, and Figure 4 is a process diagram of the mutagen testing method according to the conventional method. .
Claims (6)
反応させ、この反応により生成する酵素の活性を光学的
方法により測定して、変異原物質の定性および定量を行
なうことを特徴とする変異原物質試験方法。(1) The mutagenicity test method is characterized in that the mutagen is reacted with bacteria, and the activity of the enzyme produced by this reaction is measured by an optical method to qualitatively and quantitatively quantify the mutagen. mutagen test method.
求の範囲第１項に記載の試験方法。(2) The test method according to claim 1, wherein the mutagenicity test method is the umu test method.
ゼン（ＴＮＣＢ）、４−ニトロキノリン−Ｎ−オキシド
（４ＮＱＯ）、１，３，６，８−テトラニトロカルバゾ
ール（ＴＮＣＺ）、２−ニトロフルオレン（２ＮＦ）、
２，７−ジニトロフルオレン（ＤＮＦ）、２−ニトロ−
９−フルオレノン（２ＮＦＯ）、２，７−ジニトロ−９
−フルオレノン（ＤＮＦＯ）、２，４，７−トリニトロ
−９−フルオレノン（ＴｒｉＮＦＯ）、２，４，５，７
−テトラニトロ−９−フルオレノン（ＴｅｔｒａＮＦＯ
）およびニトロフミン酸のベンゼン−エタノール抽出物
（ＮＦＡ）の群から選ばれた少なくとも１種である特許
請求の範囲第１項に記載の試験方法。(3) The mutagen is 2,4,6-trinitrochlorobenzene (TNCB), 4-nitroquinoline-N-oxide (4NQO), 1,3,6,8-tetranitrocarbazole (TNCZ), 2- Nitrofluorene (2NF),
2,7-dinitrofluorene (DNF), 2-nitro-
9-fluorenone (2NFO), 2,7-dinitro-9
-Fluorenone (DNFO), 2,4,7-trinitro-9-fluorenone (TriNFO), 2,4,5,7
-Tetranitro-9-fluorenone (TetraNFO
) and a benzene-ethanol extract of nitrofumic acid (NFA).
である特許請求の範囲第１項に記載の試験方法。(4) The bacteria is Salmonella enterica TA1535/pSK1002
The test method according to claim 1.
特許請求の範囲第１項に記載の試験方法。(5) The test method according to claim 1, wherein the enzyme produced is β-D-galactosidase.
１項に記載の試験方法。(6) The test method according to claim 1, wherein the optical method is a chemiluminescence method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62122561A JPS63287490A (en) | 1987-05-21 | 1987-05-21 | Method for testing mutagen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62122561A JPS63287490A (en) | 1987-05-21 | 1987-05-21 | Method for testing mutagen |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS63287490A true JPS63287490A (en) | 1988-11-24 |
Family
ID=14838938
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62122561A Pending JPS63287490A (en) | 1987-05-21 | 1987-05-21 | Method for testing mutagen |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63287490A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6192887B1 (en) * | 1998-05-19 | 2001-02-27 | The Pennsylvania State University | Broad spectrum microbicidal and spermicidal compositions and methods having activity against sexually transmitted agents including papillomaviruses |
-
1987
- 1987-05-21 JP JP62122561A patent/JPS63287490A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6192887B1 (en) * | 1998-05-19 | 2001-02-27 | The Pennsylvania State University | Broad spectrum microbicidal and spermicidal compositions and methods having activity against sexually transmitted agents including papillomaviruses |
| US6458346B1 (en) | 1998-05-19 | 2002-10-01 | The Pennsylvania State University | Broad spectrum microbicidal and spermicidal compositions, devices, and methods |
| US6635242B2 (en) | 1998-05-19 | 2003-10-21 | The Pennsylvania State University | Broad spectrum microbicidal and spermicidal compositions, devices, and methods |
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