Carlson Erik S, Upadhyaya Pramod, Hecht Stephen S
Department of Pharmacology, University of Minnesota; Masonic Cancer Center, University of Minnesota;
Masonic Cancer Center, University of Minnesota.
J Vis Exp. 2017 Sep 25(127):56312. doi: 10.3791/56312.
N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted mechanism of metabolic activation involves formation of α-hydroxynitrosamines that spontaneously decompose to DNA alkylating agents. Accumulation of DNA damage and the resulting mutations can ultimately lead to cancer. New evidence indicates that α-hydroxynitrosamines can be further oxidized to nitrosamides processively by cytochrome P450s. Because nitrosamides are generally more stable than α-hydroxynitrosamines and can also alkylate DNA, nitrosamides may play a role in carcinogenesis. In this report, we describe a general protocol for evaluating nitrosamide production from in vitro cytochrome P450-catalyzed metabolism of nitrosamines. This protocol utilizes a general approach to the synthesis of the relevant nitrosamides and an in vitro cytochrome P450 metabolism assay using liquid chromatography-nanospray ionization-high resolution tandem mass spectrometry for detection. This method detected N'-nitrosonorcotinine as a minor metabolite of N'-nitrosonornicotine in the example study. The method has high sensitivity and selectively due to accurate mass detection. Application of this method to a wide variety of nitrosamine-cytochrome P450 systems will help determine the generality of this transformation. Because cytochrome P450s are polymorphic and vary in activity, a better understanding of nitrosamide formation could aid in individual cancer risk assessment.
N-亚硝胺是一类公认的环境致癌物，需要细胞色素P450氧化才能表现出活性。公认的代谢活化机制涉及α-羟基亚硝胺的形成，其可自发分解为DNA烷基化剂。DNA损伤的积累及由此产生的突变最终可导致癌症。新证据表明，α-羟基亚硝胺可被细胞色素P450进一步连续氧化为亚硝酰胺。由于亚硝酰胺通常比α-羟基亚硝胺更稳定，且也能使DNA烷基化，因此亚硝酰胺可能在致癌过程中发挥作用。在本报告中，我们描述了一种评估亚硝胺在体外经细胞色素P450催化代谢生成亚硝酰胺的通用方案。该方案采用了合成相关亚硝酰胺的通用方法以及使用液相色谱-纳米喷雾电离-高分辨率串联质谱进行检测的体外细胞色素P450代谢测定法。在示例研究中，该方法检测到N'-亚硝基去甲可替宁是N'-亚硝基去甲烟碱的一种次要代谢产物。由于精确的质量检测，该方法具有高灵敏度和选择性。将此方法应用于多种亚硝胺-细胞色素P450系统将有助于确定这种转化的普遍性。由于细胞色素P450具有多态性且活性各异，更好地了解亚硝酰胺的形成有助于进行个体癌症风险评估。
