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

Summary

α-hydroxylation of carcinogenic nitrosamines by cytochrome P450s is the accepted metabolic pathway that produces DNA-damaging intermediates, which cause mutations. However, new data indicates further oxidation to nitrosamides can occur. We describe a general method for detecting nitrosamides produced from in vitro cytochrome P450-catalyzed metabolism of nitrosamines.

Abstract

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.

Introduction

N-nitrosamines are a large class of carcinogens found in the diet, tobacco products, and the general environment; they can also be formed endogenously in the human body¹. More than 300 N-nitroso compounds have been tested and >90% were evaluated as carcinogenic in animal models^2,3. To exhibit their carcinogenicity, these compounds must first be activated by cytochrome P450s^1,2,3. Research shows that cytochrome P450s readily oxidize nitrosamines to α-hydroxynitrosamines (

Protocol

1. Materials and general procedures

1. Synthesize NNN as previously described¹⁵. Obtain norcotinine, P450 2A6 Baculosomes, NADPH regeneration system, 0.5x reaction buffer, and all other chemicals or solvents from commercial sources in reagent grade.
2. Record NMR spectra on a 500 MHz spectrometer. Report chemical shifts as parts per million (ppm). Use residual solvent peaks as internal references for ¹H-NMR (7.26 ppm CDCl₃) and ¹³C-NMR (77.2 ppm CDCl₃).
   NOTE: Peak splitting used the following abbreviations: s = singlet, d = doublet, dd = doublet of doublets, dt = doublet of....

Results

Based on the work of White et al.¹⁹, norcotinine was nitrosated to NNC cleanly and in high yield (80 - 92%) to produce a standard for the in vitro experiment. Structural evidence for a successful reaction was obtained from spectroscopic analyses including ¹H-NMR, ¹³C-NMR, COSY, and HSQC (Supporting Information) along with HRMS which confirmed the parent mass [M + H]⁺ within 5 ppm of the theoretical value (

Discussion

Elucidating the metabolism of nitrosamines is a critical component to understanding their carcinogenicity. Since the involved cytochrome P450s and other metabolic enzymes are polymorphic, further application of this knowledge could potentially identify high risk individuals^1,4. New data indicates that further oxidation of α-hydroxynitrosamines, the presumed major metabolites of nitrosamines involved in DNA binding, to nitrosamides is possible; however, this .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Norcotinine	AKoS GmbH (Steinen, Germany)	CAS 17708-87-1, AKoS AK0S006278969
Acetic acid	Sigma-Aldrich	695092
Acetic Anhydride	Sigma-Aldrich	242845
Ammonium Acetate	Sigma-Aldrich	431311
Barium Hydroxide	Sigma-Aldrich	433373
D-Chloroform	Sigma-Aldrich	151823
HPLC Acetonitrile	Sigma-Aldrich	34998
Magnesium Sulfate	Sigma-Aldrich	M7506
Methylene Chloride	Sigma-Aldrich	34856
Sodium Nitrite	Sigma-Aldrich	237213
ViVid CYP2A6 Blue Screening Kit	Life Technologies	PV6140
Zinc Sulfate	Sigma-Aldrich	221376
0.5 mL tubes	Fisher	AB0533
100 mL round bottom flask	Sigma-Aldrich	Z510424
125 mL Erlenmeyer flask	Sigma-Aldrich	CLS4980125
125 mL Separatory Funnel	Sigma-Aldrich	Z261017
25 mL round bottom flask	Sigma-Aldrich	Z278262
500 MHz NMR Spectrometer	Bruker
Allegra X-22R Centrifuge	Beckman-Coulter
LC vials	ChromTech	CTC–0957–BOND
LTQ Orbitrap Velos	Thermo Scientific
Magnetic Stir bar	Sigma-Aldrich	Z127035
NMR tube	Sigma-Aldrich	Z274682
P1000, P200, and P10 pipettes	Eppendorf
Rotary evaporator	Sigma-Aldrich	Z691410
RSLCnano UPLC system	Thermo Scientific
Shaking Water Bath	Fisher	FSSWB15
Stir plate	Sigma-Aldrich	CLS6795420
PicoFrit Column	New Objective	PF3607515N5
Luna C18, 5 um	Phenomenex	535913-1