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

TY - JOUR

T1 - Determination of genomic N3-methylthymidine in human cancer cells treated with nitrosamines using capillary electrophoresis with laser-induced fluorescence

AU - Krais, Annette M

AU - Kliem, Christian

AU - Arlt, Volker M

AU - Schmeiser, Heinz H

N1 - © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PY - 2019/6/1

Y1 - 2019/6/1

N2 - 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.

AB - 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.

KW - 5-hydroxymethylcytosine

KW - Capillary electrophoresis with laser-induced fluorescence

KW - DNA methylation

KW - N3-methylthymidine

KW - Nitrosamines

UR - http://www.scopus.com/inward/record.url?scp=85062329710&partnerID=8YFLogxK

U2 - 10.1002/elps.201800495

DO - 10.1002/elps.201800495

M3 - Article

C2 - 30767246

SN - 0173-0835

VL - 40

SP - 1535

EP - 1539

JO - Electrophoresis

JF - Electrophoresis

IS - 11

ER -