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

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

doi:10.1007/s00204-018-2355-0

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.

Highlights

Genotoxic N-nitroso compounds (NOC) are widespread in our diet and environment
The antibody has been successfully employed for immuno-slot blot (ISB) analysis of O6-MeG lesions in genomic DNA extracted from cells and tissue (Fahrer et al 2015; Mikhed et al 2016; Stephanou et al 1996), and an ELISA-related assay, in which single-stranded DNA fragments with O6-MeG were captured by the antibody followed by the detection of the ssDNA (Georgiadis et al 2011)
In this study, levels of O6-MeG resulting from exposure to either the anticancer drug TMZ or the colonotropic carcinogen AOM were evaluated in colorectal cancer (CRC) cells and different murine tissue by a combination of qualitative immunological assays and quantitative mass spectrometry (Fig. 1A)

Summary

Introduction

Genotoxic N-nitroso compounds (NOC) are widespread in our diet and environment. They are found in processed meat and fish, beer, cosmetics and cigarette smoke (Fahrer and Kaina 2013). NOC are formed endogenously in the stomach and the large intestine by nitrosation of amino acids (Fahrer and Kaina 2013) These critical compounds cause DNA alkylation and thereby generate a plethora of DNA adducts, such as Nmethylpurines and O6-methylguanine (O6-MeG) (Fu et al 2012). Alkylating agents play an important role in cancer chemotherapy due to their induction of cytotoxic DSBs, resulting mainly from subsequent replications over O6-MeG These methylating anticancer drugs include temozolomide (TMZ) and dacarbazine (DTIC), which are both triazine compounds used for the treatment of high-grade glioma and malignant melanoma (Kaina et al 2010). Using hockey-stick dose-response modeling, our data revealed, for the first time, a genotoxic threshold for O6-MeG formation in vivo, resulting from MGMT repair activity

Methods

Results

Conclusion