Immunological and mass spectrometry-based approaches to determine thresholds of the mutagenic DNA adduct O6-methylguanine in vivo

Abstract

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.

Keywords: Alkylating agents; O 6-methylguanine; O 6-methylguanine-DNA methyltransferase (MGMT); Thresholds; Ultra performance liquid chromatography–tandem mass spectrometry.