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Determination of genomic N3-methylthymidine in human cancer cells treated with nitrosamines using capillary electrophoresis with laser-induced fluorescence

Research output: Contribution to journalArticle

Annette M Krais, Christian Kliem, Volker M Arlt, Heinz H Schmeiser

Original languageEnglish
Pages (from-to)1535-1539
Number of pages5
JournalElectrophoresis
Volume40
Issue number11
Early online date15 Feb 2019
DOIs
Accepted/In press3 Feb 2019
E-pub ahead of print15 Feb 2019
Published1 Jun 2019

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© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Abstract

Methylating substances alter DNA by forming N3-methylthymidine (N3mT), a mutagenic base modification. To develop a sensitive analytical method for the detection of N3mT in DNA based on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF), we synthesized the N3mT-3'-phosphate as a chemical standard. The limit of detection was 1.9 amol of N3mT, which corresponds to one molecule of N3mT per 1,000 normal nucleotides or 0.1%. With this method, we demonstrated that the carcinogenic nitrosamine N'-nitrosonornicotine (NNN) induced N3mT in the human lung cancer cell line A549. Treatment with NNN also caused an elevated degree of 5-hydroxymethylcytidine (5hmdC) in DNA, while the methylation degree (i.e. 5-methylcytidine; 5mdC) stayed constant. According to our data, NNN could, via yet unknown mechanisms, play a role in the formation of N3mT as well as 5hmdC. In this study we have developed a new sensitive analytical method using CE-LIF for the simultaneous detection of the three DNA modifications, 5mdC, 5hmdC and N3mT. This article is protected by copyright. All rights reserved.

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