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Supernucleophilic Cob(I)alamin: analytical tool for genotoxic compounds in vitro and in vivo
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The low but still non-acceptable levels of exposure to genotoxic compounds emphasises the need for sensitive methods for searching, detecting, identifying and quantifying these compounds. Within the field of studies on genotoxic compounds, chemically quantitative work is required throughout.

This work comprises the development of a method for analysis of DNA-phosphate adducts formed after exposure to genotoxic compounds, i.e. compounds which are electrophiles. The possibility of utilising the weakly alkylating property of the phosphotriester configuration by transferring the alkyl groups (adducts) to a strong nucleophile has been demonstrated using model phosphotriesters. Standard nucleophiles were shown to perform nucleophilic displacement on the alkyl group in a phosphotriester configuration with the formation of an alkyl-nucleophile complex suitable for analysis. However, reaction-kinetic studies demonstrated too low rates of the transalkylation reaction for practical use. Therefore, the supernucleophilic cob(I)alamin was introduced and tested as a nucleophile for transalkylation. It was shown to perform the same transalkylation reaction 4,000 times faster than the tested standard nucleophiles. The transalkylation method was further developed for applicability to DNA. Degradation of DNA by nucleolytic enzymes and phosphatase resulting in phosphotriester products was introduced prior to the transalkylation procedure. The transalkylation method was applied to the determination of phosphate adducts in DNA from mice treated with the tobacco-specific nitrosamine, NNK, and the existence of phosphate adducts was demonstrated. In addition, the adapted degradation method was applied to DNA from mice treated with benzo[a]pyrene for the determination of N2-guanine adducts.

The high nucleophilic strength of cob(I)alamin was a reason for further investigation of its usefulness as an analytical tool. It was applied to reaction-kinetic studies of oxiranes, and was shown to be a superior nucleophile for trapping and analysis of electrophilically reactive compounds. In applications utilizing cobalamin for solving different toxicological problems a mass spectrometric method permitting identification and quantification of formed alkyl-cobalamins with high sensitivity is required. An electrospray LC/MS/MS method fulfilling these requirements was developed. To sum up, the supernucleophilic cob(I)alamin has in this study been shown to provide a superior analytical tool for identification and quantification of genotoxic compounds in vitro and in vivo

Place, publisher, year, edition, pages
Stockholm: Stockholm University , 2001. , 67 p.
National Category
Biological Sciences
Research subject
Genetic Toxicology
Identifiers
URN: urn:nbn:se:su:diva-143002ISBN: 91-7265-337-X (print)OAI: oai:DiVA.org:su-143002DiVA: diva2:1094080
Public defence
2001-09-28, 10:00 (English)
Opponent
Note

Härtill 7 uppsatser

Available from: 2017-05-09 Created: 2017-05-09 Last updated: 2017-09-28Bibliographically approved

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Citation style
  • apa
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