A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s.

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Journal of visualized experiments : JoVE, ISSN: 1940-087X, Vol: 2017, Issue: 127, Page: e56312-e56312

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Erik S. Carlson, Pramod Upadhyaya, Stephen S. Hecht
MyJove Corporation
Neuroscience, Chemical Engineering, Biochemistry, Genetics and Molecular Biology, Immunology and Microbiology
article description
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.

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