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Immunological and mass spectrometry-based approaches to determine thresholds of the mutagenic DNA adduct O 6 -methylguanine in vivo

Kraus, Alexander; McKeague, Maureen; Seiwert, Nina; Nagel, Georg; Geisen, Susanne M.; Ziegler, Nathalie; Trantakis, Ioannis A.; Kaina, Bernd; Thomas, Adam D.; Sturla, Shana J.; Fahrer, Jörg

Immunological and mass spectrometry-based approaches to determine thresholds of the mutagenic DNA adduct O 6 -methylguanine in vivo Thumbnail


Authors

Alexander Kraus

Maureen McKeague

Nina Seiwert

Georg Nagel

Susanne M. Geisen

Nathalie Ziegler

Ioannis A. Trantakis

Bernd Kaina

Adam Thomas Adam7.Thomas@uwe.ac.uk
Senior Lecturer in Human Genetics and Genomics

Shana J. Sturla

Jörg Fahrer



Abstract

© 2018, Springer-Verlag GmbH Germany, part of Springer Nature. N-nitroso compounds are alkylating agents, which are widespread in our diet and the environment. They induce DNA alkylation adducts such as O 6 -methylguanine (O 6 -MeG), which is repaired by O 6 -methylguanine-DNA methyltransferase (MGMT). Persistent O 6 -MeG lesions have detrimental biological consequences like mutagenicity and cytotoxicity. Due to its pivotal role in the etiology of cancer and in cytotoxic cancer therapy, it is important to detect and quantify O 6 -MeG in biological specimens in a sensitive and accurate manner. Here, we used immunological approaches and established an ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) to monitor O 6 -MeG adducts. First, colorectal cancer (CRC) cells were treated with the methylating anticancer drug temozolomide (TMZ). Immunofluorescence microscopy and an immuno-slot blot assay, both based on an adduct-specific antibody, allowed for the semi-quantitative, dose-dependent assessment of O 6 -MeG in CRC cells. Using the highly sensitive and specific UPLC–MS/MS, TMZ-induced O 6 -MeG adducts were quantified in CRC cells and even in peripheral blood mononuclear cells exposed to clinically relevant TMZ doses. Furthermore, all methodologies were used to detect O 6 -MeG in wildtype (WT) and MGMT-deficient mice challenged with the carcinogen azoxymethane. UPLC–MS/MS measurements and dose–response modeling revealed a non-linear formation of hepatic and colonic O 6 -MeG adducts in WT, whereas linear O 6 -MeG formation without a threshold was observed in MGMT-deficient mice. Collectively, the UPLC–MS/MS analysis is highly sensitive and specific for O 6 -MeG, thereby allowing for the first time for the determination of a genotoxic threshold upon exposure to O 6 -methylating agents. We envision that this method will be instrumental to monitor the efficacy of methylating chemotherapy and to assess dietary exposures.

Citation

Kraus, A., McKeague, M., Seiwert, N., Nagel, G., Geisen, S. M., Ziegler, N., …Fahrer, J. (2019). Immunological and mass spectrometry-based approaches to determine thresholds of the mutagenic DNA adduct O 6 -methylguanine in vivo. Archives of Toxicology, 93(2), 559-572. https://doi.org/10.1007/s00204-018-2355-0

Journal Article Type Article
Acceptance Date Nov 8, 2018
Online Publication Date Nov 16, 2018
Publication Date Feb 6, 2019
Publicly Available Date Nov 17, 2019
Journal Archives of Toxicology
Print ISSN 0340-5761
Electronic ISSN 1432-0738
Publisher Springer (part of Springer Nature)
Peer Reviewed Peer Reviewed
Volume 93
Issue 2
Pages 559-572
DOI https://doi.org/10.1007/s00204-018-2355-0
Keywords O6-methylguanine, O6-methylguanine-DNA methyltransferase (MGMT), alkylating agents, ultra performance liquid chromatography–tandem mass spectrometry, thresholds
Public URL https://uwe-repository.worktribe.com/output/851524
Publisher URL https://doi.org/10.1007/s00204-018-2355-0
Additional Information Additional Information : This is a post-peer-review, pre-copyedit version of an article published in Archives of Toxicology. The final authenticated version is available online at: https://doi.org/10.1007/s00204-018-2355-0

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