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Dection of strand breaks in φX 174 RFI and PM2 DNA reacted with ultimate and proximate carcinogens

Nachweis von Strangbrüchen in φX RFI und PM2 DNA nach Umsetzung mit direkten und indirekten Carcinogenen

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Summary

Supercoiled DNA duplexes of phages φX 174 and PM2 were treated in aqueous solution at neutral pH with ultimate and proximate carcinogens. Subsequently, the carcinogen-treated phage DNAs were subjected to velocity sedimentation in neutral and alkaline sucrose to quantitate introduction of single strand breaks.

Reaction of phage DNA with the ultimate carcinogens N-methyl-N-nitrosourea (MeNOUr), N-ethyl-N-nitrosourea (EtNOUr), 7-bromomethyl-benz[a]-anthracene, N-acetoxy-2-acetylaminofluorene [(Ac)2ONFln] and K-region oxides for short periods followed by sedimentation in neutral sucrose gradients led to very few breaks. Incubation with the proximate carcinogens N-hydroxy-2-acetylaminofluorene, 2-acetylaminofluorene, 7-methyl-, and 7,12-dimethyl-benz[a]anthracene did not result in breaks.

However, when the phage DNAs were reacted with the ultimate carcinogens under the same conditions but subsequently alkali-denatured and sedimented in alkaline sucrose gradients, single strand breaks were readily introduced. Incubation with the proximate carcinogens followed by alkali denaturation and sedimentation in alkaline sucrose showed that only 7,12-dimethylbenz[a]anthracene and, to a minor extent, 7-methyl-benz[a]anthracene caused alkali-inducible breaks.

The ability of N-methyl-N′-nitro-N-nitrosoguanidine to effect breakdown of superhelical phage DNA in alkali was found enhanced in the presence of N-acetyl-cysteine.

Zusammenfassung

Superhelikale doppelsträngige DNA der Phagen φX 174 und PM2 wurde in wäßriger Lösung bei neutralem pH mit direkten und indirekten Carcinogenen umgesetzt. Anschließend wurde die Carcinogen-behandelte DNA einer Geschwindigkeitssedimentation in neutraler und alkalischer Sucrose unterworfen, um Einzelstrangbrüche quantitativ zu erfassen. Kurzzeitige Reaktion der Phagen DNA mit den direkten Carcinogenen N-Methyl-N-nitrosoharnstoff (MeNOUr), N-Äthyl-N-nitrosoharnstoff (EtNOUr), 7-Brommethyl-benz[a]anthracen, N-Acetoxy-2-acetylaminofluoren [(Ac)2ONFln] und K-Region Oxiden, gefolgt von Sedimentation in neutralen Sucrosegradienten, führte zu nur wenigen Brüchen. Nach Inkubation mit den indirekten Carcinogenen N-Hydroxy-2-acetylaminofluoren, 2-Acetylaminofluoren, 7-Methyl-und 7,12-Dimethyl-benz[a]anthracen wurden keine Brüche beobachtet.

Wurde dagegen die Phagen-DNA mit den direkten Carcinogenen unter denselben Reaktionsbedingungen umgesetzt, jedoch anschließend mit Alkali denaturiert und in alkalischen Sucrosegradienten sedimentiert, so waren zahlreiche Einzelstrangbrüche nachweisbar.

Die Inkubation der DNA mit indirekten Carcinogenen und die Analyse der Produkte in alkalischen Sucrosegradienten ergab, daß nur 7,12-Dimethylbenz[a]anthracen und, in geringem Umfang, 7-Methyl-benz[a]anthracen zu Brüchen führten.

N-Methyl-N′-nitro-N-nitrosoguanidin bewirkte alkali-katalysierbare Brüche in superhelikaler Phagen-DNA; dieser Effekt war besonders ausgeprägt in Gegenwart von N-Acetyl-cystein.

In weiteren Experimenten wurde PM2 DNA mit abgestuften Konzentrationen von [3H]MeNOUr, 7-Brom[14C]methyl-benz[a]anthracen und N-Acetoxy-2-[2-3H]acetylaminofluoren umgesetzt. Durch gleichzeitige Sedimentationsanalyse in neutraler und alkalischer Sucrose wurden die Anzahl der pro PM2 Duplex kovalent gebundenen Carcinogen-Reste sowie die Anzahl alkalikatalysierter Brüche bestimmt. Auf den gleichen Betrag gebundener Carcinogen-Reste bezogen, nimmt die Fähigkeit dieser Carcinogene, alkali-empfindliche Stellen in DNA einzuführen, in der Reihenfolge: 7-Brommethylbenz[a]anthracen>MeNOUr>(Ac)2ONFln ab. MeNOUr und EtNOUr bilden in DNA Alkyl-phosphotriester; weiterhin alkylieren sie unter anderem N-7 und O6 des Guanins sowie N-3 des Adenins. Diese Basenmodifikation führt zur Labilisierung der jeweiligen N-glykosidischen Bindung und zu Basenverlust (Apurinstelle). Sowohl Apurinstellen als auch Alkyl-phosphotriester sind alkali-empfindlich. Es ist wahrscheinlich, daß 7-Methyl-und 7,12-Dimethylbenz[a]anthracen (beide möglicherweise durch Luftsauerstoff aktiviert) sowie 7-Brommethyl-benz[a]anthracen, (Ac)2ONFln und die getesteten K-Region Oxide ihre Wirkung aufgrund derselben Mechanismen entfalten.

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Abbreviations

Me2SO:

dimethyl sulfoxide

MeNOUr:

N-methyl-N-nitrosourea

EtNOUr:

N-ethyl-N-nitrosourea

(Ac)2ONFln:

N-acetoxy-2-acetylaminofluorene

(Ac)NOHFln:

W-hydroxy-2-acetylaminofluorene

(Ac)NHFln:

2-acetylaminofluorene

Me(NO)(NO2)Gdn:

N-methyl-N′-nitro-N-nitrosoguanidine

RF:

double-stranded replicative form DNA

RF I:

the supertwistet DNA duplex

RF II:

the open-stranded DNA duplex

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Authors and Affiliations

  1. Institut für Biochemie, Deutsches Krebsforschungszentrum, Heidelberg, Federal Republic of Germany

    H. W. Thielmann

  2. Abteilung für Molekulare Biologie, Max-Planck-Institut für Medizinische Forschung, Im Neuenheimer Feld 280, D-6900, Heidelberg, Federal Republic of Germany

    H. W. Thielmann

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This work was supported by the Deutsche Forschungsgemeinschaft

I thank Prof. E. Hecker for stimulating discussions during the experimental work. I am very grateful to Miss U. Reygers and Mr. H. Gersbach for excellent technical assistance. I also thank R. S. Schmidt and Dr. Howard V. Hershey for critically reading the manuscript and helpful suggestions.

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Thielmann, H.W. Dection of strand breaks in φX 174 RFI and PM2 DNA reacted with ultimate and proximate carcinogens. Z. Krebsforsch. 90, 37–69 (1977). https://doi.org/10.1007/BF00306020

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