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首页> 外文期刊>Archives of Toxicology >Immunological and mass spectrometry-based approaches to determine thresholds of the mutagenic DNA adduct O-6-methylguanine in vivo
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Immunological and mass spectrometry-based approaches to determine thresholds of the mutagenic DNA adduct O-6-methylguanine in vivo

机译：基于免疫和质谱的方法，以确定体内诱变DNA加合物O-6-甲基胍的阈值

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摘要

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.
机译：N-亚硝基化合物是烷基化试剂，其在我们的饮食和环境中普遍存在。它们诱导DNA烷基化加合物，例如O-6-甲基庚烷（O-6-MEG），其由O-6-甲基胍-DNA甲基转移酶（MGMT）修复。持续的O-6-MEG病变具有诱变和细胞毒性等生物后果。由于其在癌症的病因和细胞毒性癌症治疗中的关键作用，重要的是以敏感和准确的方式检测和量化生物标本中的O-6-MEG。在此，我们使用免疫学方法并建立了超高效液相色谱 - 串联质谱（UPLC-MS / MS）以监测O-6-MEG加合物。首先，用甲基化抗癌药物替莫唑胺（TMZ）处理结直肠癌（CRC）细胞。免疫荧光显微镜和免疫槽印迹测定基于加合物特异性抗体，允许对CRC细胞中O-6-Meg的半定量，剂量依赖性评估。使用高敏感和特异性UPLC-MS / MS，TMZ诱导的O-6-MEG加合物在CRC细胞中量化，即使在暴露于临床相关的TMZ剂量的外周血单核细胞中。此外，所有方法都用于检测野生型（WT）的O-6-MEG，并用致癌甲氧基甲烷攻击的MGMT缺陷小鼠。 UPLC-MS / MS测量和剂量 - 响应建模揭示了WT中的肝脏和结肠O-6-MEG加合物的非线性形成，而在MGMT缺陷小鼠中观察到没有阈值的线性O-6-MEG形成。集体，UPLC-MS / MS分析对O-6-MEG的高度敏感和特异性，从而允许在暴露于O-6-甲基化试剂时测定基因毒性阈值。我们想象这种方法将有助于监测甲基化化学疗法的功效并评估膳食曝光。

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