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, J�rg

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.

• Journal Article Type: Article
• Acceptance Date: Nov 8, 2018
• Online Publication Date: Nov 16, 2018
• Publication Date: Feb 6, 2019
• Deposit Date: Nov 21, 2018
• 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
• Contract Date: Nov 26, 2018