Highly sensitive sites for guanine-O6 ethylation in rat brain DNA exposed to N-ethyl-N-nitrosourea in vivo.

Nehls, Peter; Rajewsky, Manfred F.; Spieß, Eberhard; Werner, Dieter

doi:10.1002/j.1460-2075.1984.tb01806.x

Cited by 43 publications

(16 citation statements)

References 21 publications

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“…In general, the cellular content of DNA lesions depends on two counteracting processes: a) the amount and ability of an ultimate carcinogen to react with the constituents of genomic DNA that are packed in chromatin in situ (25,46), and b) the ability of a cell to recognize and eliminate the lesions from DNA by repair proteins. Formation of large numbers of DNA lesions and/or an inefficient cellular repair activity lead to increased levels (persistence) of DNA lesions.…”

Section: Discussionmentioning

confidence: 99%

“…These mechanisms comprise antioxidant systems for the deactivation of ROS molecules and efficient repair proteins for the elimination of oxidative DNA damage (19)(20)(21)(22)(23) (24)(25)(26)(27). These techniques can be applied for the determination of any DNA modification for which an appropriate monoclonal or polyclonal antibody is available.…”

Section: Introductionmentioning

confidence: 99%

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Formation and persistence of 8-oxoguanine in rat lung cells as an important determinant for tumor formation following particle exposure.

Nehls

Seiler

Rehn

et al. 1997

Environ Health Perspect

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Exposure of rats to quartz (or various other particles) can lead to the development of lung tumors. At the moment, the mechanisms involved in particle-induced tumor formation are not clarified. However, it is suggested that inflammation, in conjunction with the production of reactive oxygen species (ROS) and an enhancement of epithelial cell proliferation, may play a key role in the development of lung tumors. ROS induces 8-oxoguanine (8-oxoGua) and other mutagenic DNA oxidation products, which can be converted to mutations in proliferating cells. Mutation formation in cancer-related genes is a critical event with respect to tumor formation. In this study we investigated the effects of quartz (DQ1 2) and of the nontumorigenic dust corundum on the induction of 8-oxoGua in the DNA of rat lung cells, as well as on cell proliferation and pulmonary inflammation. Wistar rats were exposed by intratracheal instillation to quartz (2.5 mg/rat) or corundum (2.5 mg/rat) suspended in physiological saline; control animals exposed to physiological saline or left untreated. Measurements were carried out 7, 21, and 90 days after the exposures. 8-oxoGua levels were determined in lung tissue sections at the single cell level by immunocytological assay using a rabbit anti-8-oxoGua antibody. After exposure to quartz, 8-oxoGua levels were significantly increased at all time points of investigation. Additionally, we observed inflammation and an enhanced cell proliferation. Exposure to corundum had no adverse effects on the lung; neither increased 8-oxoGua levels nor enhanced cell proliferation or inflammation were detected. These observations support the suggestion that inflammation associated with increased 8-oxoGua levels in lung cells and increased cell proliferation is an important determinant for particle-induced development of lung tumors in the rat.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Formation and persistence of 8-oxoguanine in rat lung cells as an important determinant for tumor formation following particle exposure.

Nehls

Seiler

Rehn

et al. 1997

Environ Health Perspect

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“…With the use of immuno-electron microscopy (IEM) in conjunction with a protein-free DNA spreading technique (25), specific alkyldeoxynucleosides can be visualized in double-stranded DNA molecules via Mabbinding sites. In a typical preparation for IEM of DNA containing 06-EtdGuo, and anti-(O6-EtdGuo) Mab (e.g., Mab ER-6; 5, 25; 80 ,ug/mL (is incubated with DNA (50 ,ug/mL) in TMS-buffer (10 mM triethanolamine, 100 mM Mg-acetate, 150 mM NaCl, 10 mM EDTA, 0.02% NaN3, pH 7.2).…”

Section: Immuno-electron Microscopy (Iem)mentioning

confidence: 99%

“…While other laboratories have focused on the production of antisera, and in some cases of Mab, directed against DNA components structurally altered by reaction with aflatoxin B1, N-2-acetylaminofluorene, or benzo(a)pyrene (7,9,(12)(13)(14)(15), our group has concentrated on the development of Mab specific for alkyldeoxynucleosides produced in cellular DNA by alkylating Nnitroso compounds (5,6,11,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). We have thus exploited the exceptional capability of antibodies to recognize subtle alterations of molecular structure, in order to distinguish deoxynucleosides modified by the covalent attachment of single, small alkyl groups (methyl, ethyl, butyl, isopropyl) from their normal, unaltered counterparts.…”

Section: Introductionmentioning

confidence: 99%

“…Mab with very high affinity and specificity for the respective alkyl-deoxynucleosides were obtained by immunization with alkylribonucleosides as haptens coupled to keyhole limpet hemocyanin (KLH) as a carrier protein (5,6,22). With the use of different immunoanalytical methods, specific alkyldeoxynucleosides can now be quantitated with high sensitivity in small amounts of DNA or in DNA hydrolyzed to monodeoxynucleosides, in the DNA of individual cells, and in single DNA molecules (19,21,22,(24)(25)(26). In the present report, we describe the properties of Mab specific for the DNA alkylation products 06-methyldeoxyguanosine (06-MedGuo), O6-ethyldeoxyguanosine (06-EtdGuo), 06-butyldeoxyguanosine (06-BudGuo), 06-isopropyldeoxyguanosine (06-iProdGuo), 04-methyldeoxythymidine (04-MedThd), and 04-ethyldeoxythymidine (04-EtdThd).…”

Section: Introductionmentioning

confidence: 99%

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Quantitation and visualization of alkyl deoxynucleosides in the DNA of mammalian cells by monoclonal antibodies.

Adamkiewicz¹,

Eberle²,

Huh³

et al. 1985

Environ Health Perspect

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Conventional radiochromatographic procedures for the quantitation of carcinogen/mutagen-induced structural DNA modifications have a number of limitations. Thus, these techniques for the most part require application of radioactively labeled carcinogens and the use of relatively large amounts of DNA for analysis at low levels of DNA modification. Radiochromatographic methods also preclude analyses at the level of single cells and DNA molecules. Recently developed immunoanalytical methods have improved this situation considerably. Monoclonal antibodies (Mab) characterized by a high substrate specificity and affinity, in combination with radio-and enzyme-immunoassays, or with "immuno-slot-blot" techniques, now permit the detection of femtomole to subfemtomole amounts of, e.g., alkyldeoxynucleosides in small samples of DNA isolated from tissues or cultured cells previously exposed to nonradioactive N-nitroso compounds. Furthermore, selected Mab can be used to quantitate by direct immunofluorescence (with the aid of computer-based image analysis of electronically intensified fluorescence signals), specific alkyldeoxynucleosides in the nuclear DNA of single cells. With this method, the detection limit for the alkylation product 0'-ethyldeoxyguanosine (06-EtdGuo) is presently of the order of W-103 06-EtdGuo residues per diploid mammalian genome. Individual cells can thus be monitored for the presence of specific carcinogen-DNA adducts, and with respect to their capacity for enzymatic removal of such modified structures from DNA (as exemplified here by the kinetics of the enzymatic elimination of 06-EtdGuo from the DNA of malignant neurogenic rat cell lines). In combination with transmission electron microscopy, Mab also permit direct visualization (via Mab binding sites) of specific carcinogen-modified structures in individual DNA molecules. DNA strands of defined nucleotide sequence can thus be analyzed by immuno-electron microscopy for the presence of "hot spots" of specific structural alterations caused by defined carcinogens/ mutagens.

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Platinum Nucleobase Chemistry

Lippert

1989

Progress in Inorganic Chemistry

200

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Highly sensitive sites for guanine-O6 ethylation in rat brain DNA exposed to N-ethyl-N-nitrosourea in vivo.

Abstract: corresponding to 2970 4 1760 bp) the average ABS-ABS interspace distance was 110 nm (= 390 bp; range -9-600 nm), indicating a highly non-random distribution of 06-EtdGuo in target cell DNA.

Cited by 43 publications

References 21 publications

Formation and persistence of 8-oxoguanine in rat lung cells as an important determinant for tumor formation following particle exposure.

Formation and persistence of 8-oxoguanine in rat lung cells as an important determinant for tumor formation following particle exposure.

Quantitation and visualization of alkyl deoxynucleosides in the DNA of mammalian cells by monoclonal antibodies.

Platinum Nucleobase Chemistry

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