Immunological and mass spectrometry-based approaches to determine thresholds of the mutagenic DNA adduct O6-methylguanine in vivo
N-nitroso compounds are alkylating agents, which are widespread in our diet and the environment. They induce DNA alkylation adducts such as O6-methylguanine (O6-MeG), which is repaired by O6-methylguanine-DNA methyltransferase (MGMT). Persistent O6-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 O6-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 O6-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 O6-MeG in CRC cells. Using the highly sensitive and specific UPLC–MS/MS, TMZ-induced O6-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 O6-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 O6-MeG adducts in WT, whereas linear O6-MeG formation without a threshold was observed in MGMT-deficient mice. Collectively, the UPLC–MS/MS analysis is highly sensitive and specific for O6-MeG, thereby allowing for the first time for the determination of a genotoxic threshold upon exposure to O6-methylating agents. We envision that this method will be instrumental to monitor the efficacy of methylating chemotherapy and to assess dietary exposures.
KeywordsO6-methylguanine O6-methylguanine-DNA methyltransferase (MGMT) Alkylating agents Ultra performance liquid chromatography–tandem mass spectrometry Thresholds
This work was supported by the University Medical Center Mainz (MAIFOR), the German Research Foundation (DFG-FA1034/3-1 and DFG-KA724/29-1) and the Swiss National Science Foundation (156280). We are indebted to Dr. Daniel Heylmann (Department of Toxicology, University Medical Center, Mainz, Germany) for isolating PBMCs from buffy coat. We are also grateful to Dr. Bert Vogelstein (John Hopkins University, Baltimore, USA) for providing HCT116 cells, to Dr. Leona D. Samson (MIT, Boston, USA) for providing MGMT knockout animals, and to Dr. Geoffrey P. Margison (University of Manchester, UK) for providing TMZ. We also thank the FGCZ Functional Genomics Center Zürich (FGCZ) for LC–MS/MS assistance.
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Conflict of interest
The authors declare no conflict of interest.
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