Name: a general method for detecting nitrosamide formation in the in vitro metabolism of nitrosamines by cytochrome p450s
Uploaded: 2017-09-25T13:00:00
Description: Watch this Scientific Journal Video about A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s at JoVE.com

This study was supported by grant no. CA-81301 from the National Cancer Institute. We thank Bob Carlson for editorial assistance, Dr. Peter Villalta and Xun Ming for mass spectrometry assistance in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, and Dr. Adam T. Zarth and Dr. Anna K. Michel for their valuable discussions and input. The Analytical Biochemistry Shared Resource is partially supported by National Cancer Institute Cancer Center Support Grant CA-77598

1. Materials and general procedures
<ol>
	<li>Synthesize NNN as previously described15. Obtain norcotinine, P450 2A6 Baculosomes, NADPH regeneration system, 0.5x&#160;reaction buffer, and all other chemicals or solvents from commercial sources in reagent grade.</li>
	<li>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 pp...

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+chromatographic+technique+for+the+detection+of+dye-binding.">A rapid chromatographic technique for the detection of dye-binding.</a> Anal Biochem. 89 (2), 317-323 (1978).</li><li>Pauli, G. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Chemistry+of+the+N-Alkyl-N-nitrosoamides.+I.+Methods+of+Preparation.">The Chemistry of the N-Alkyl-N-nitrosoamides. I. Methods of Preparation.</a> J. Am. Chem. Soc. 77, 6008-6010 (1955).</li><li>Patten, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+cytochrome+P450+2A6+and+3A4+as+major+catalysts+for+N'-nitrosonornicotine+alpha-hydroxylation+by+human+liver+microsomes.">Evidence for cytochrome P450 2A6 and 3A4 as major catalysts for N'-nitrosonornicotine alpha-hydroxylation by human liver microsomes.</a> Carcinogenesis. 18, 1623-1630 (1997).</li><li>Wong, H. L., Murphy, S. E., Hecht, S. 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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 individuals1,4. New data indicates that further oxidation of &#945;-hydroxynitrosamines, the presumed major metabolites of nitrosamines involved in DNA binding, to nitrosamides is possible; however, this ...

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 &#62;90% were evaluated as carcinogenic in animal models2,3. To exhibit their carcinogenicity, these compounds must first be activated by cytochrome P450s1,2,3. Research shows that cytochrome P450s readily oxidize nitrosamines to &#945;-hydroxynitrosamines (Figure 1), which are highly reactive compounds with half-lives of ~5 s before spontaneously decomposing to alkyldiazohydroxides. The latter can alkylate DNA after the loss of H2O and N2. The resulting DNA adducts, if unrepaired, can cause mutations that, if in critical onco- or tumor suppressor genes, lead to cancer development1. For this reason, much effort has been expended to acquire a full understanding of the metabolic pathways, DNA adducts, and downstream metabolites of cytochrome P450 oxidation of carcinogenic nitrosamines. This knowledge has potential application in individual cancer risk assessment4.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56312/56312fig1.jpg" /> 
Figure 1: General and proposed metabolism of nitrosamines. 
Nitrosamines (1) are oxidized by P450s to &#945;-hydroxynitrosamines (2) that spontaneously decompose to alkyldiazohydroxides (3). These compounds can bind to DNA to form DNA adducts. It is hypothesized that 2 are further oxidized by P450s to nitrosamides 4. These can directly bind to DNA to form novel DNA adducts or be hydrolyzed to 3 to form known DNA adducts. R1 and R2 represent any alkyl group. <a href="//cloudflare.jove.com/files/ftp_upload/56312/56312fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Although the &#945;-hydroxynitrosamine hypothesis is solidly supported by extensive data, there are a few inconsistencies; a major one is the short half-life of &#945;-hydroxynitrosamines5,6. It is known that these compounds are produced at the endoplasmic reticulum membrane and later alkylate nuclear DNA. Given their lifetime of a few seconds, it is puzzling how these intermediates survive the required travel though the cytosol. One hypothesis is that a portion of the &#945;-hydroxynitrosamines are processively oxidized to nitrosamides7,8, which are quite stable in comparison9. This would presumably occur via retention of &#945;-hydroxynitrosamines in the cytochrome P450 active site. Precedent for this type of oxidation has been seen with nicotine10, alcohols11, and simple alkylnitrosamines12,13. Additionally, nitrosamides are direct-acting carcinogens2,3. Based on their reactivity9, these compounds are believed to produce DNA adducts identical to those resulting from &#945;-hydroxynitrosamines along with new, unexplored DNA adducts (Figure 1). Thus, this hypothesis not only explains the transport through the cytosol, but also the formation of DNA damage products.
In this paper, a general protocol for assessing the in vitro cytochrome P450-mediated conversion of nitrosamines to nitrosamides is described. The previously reported conversion of N&#8242;-nitrosonornicotine (NNN) to N&#8242;-nitrosonorcotinine (NNC) by cytochrome P450 2A6 is showcased as an example14. Application of this protocol to a wide range of substrate-enzyme systems will help determine the importance of nitrosamides in overall nitrosamine metabolism.

<table>
	<tbody>
		<tr>
			<td>Norcotinine</td>
			<td>AKoS GmbH (Steinen, Germany)</td>
			<td>CAS 17708-87-1, AKoS AK0S006278969</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>695092</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Anhydride</td>
			<td>Sigma-Aldrich</td>
			<td>242845</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>431311</td>
			<td></td>
		</tr>
		<tr>
			<td>Barium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>433373</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>151823</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>34998</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>34856</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Nitrite</td>
			<td>Sigma-Aldrich</td>
			<td>237213</td>
			<td></td>
		</tr>
		<tr>
			<td>ViVid CYP2A6 Blue Screening Kit</td>
			<td>Life Technologies</td>
			<td>PV6140</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>221376</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL tubes</td>
			<td>Fisher</td>
			<td>AB0533</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL round bottom flask</td>
			<td>Sigma-Aldrich</td>
			<td>Z510424</td>
			<td></td>
		</tr>
		<tr>
			<td>125 mL Erlenmeyer flask</td>
			<td>Sigma-Aldrich</td>
			<td>CLS4980125</td>
			<td></td>
		</tr>
		<tr>
			<td>125 mL Separatory Funnel</td>
			<td>Sigma-Aldrich</td>
			<td>Z261017</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL round bottom flask</td>
			<td>Sigma-Aldrich</td>
			<td>Z278262</td>
			<td></td>
		</tr>
		<tr>
			<td>500 MHz NMR Spectrometer</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra X-22R Centrifuge</td>
			<td>Beckman-Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LC vials</td>
			<td>ChromTech</td>
			<td>CTC&#8211;0957&#8211;BOND</td>
			<td></td>
		</tr>
		<tr>
			<td>LTQ Orbitrap Velos</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Stir bar</td>
			<td>Sigma-Aldrich</td>
			<td>Z127035</td>
			<td></td>
		</tr>
		<tr>
			<td>NMR tube</td>
			<td>Sigma-Aldrich</td>
			<td>Z274682</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000, P200, and P10 pipettes</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>Sigma-Aldrich</td>
			<td>Z691410</td>
			<td></td>
		</tr>
		<tr>
			<td>RSLCnano UPLC system</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking Water Bath</td>
			<td>Fisher</td>
			<td>FSSWB15</td>
			<td></td>
		</tr>
		<tr>
			<td>Stir plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS6795420</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoFrit Column</td>
			<td>New Objective</td>
			<td>PF3607515N5</td>
			<td></td>
		</tr>
		<tr>
			<td>Luna C18, 5 um</td>
			<td>Phenomenex</td>
			<td>535913-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

a general method for detecting nitrosamide formation in the in vitro metabolism of nitrosamines by cytochrome p450s

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 &#945;-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 &#945;-hydroxynitrosamines can be further oxidized to nitrosamides processively by cytochrome P450s. Because nitrosamides are generally more stable than &#945;-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&#8242;-nitrosonorcotinine as a minor metabolite of N&#8242;-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.

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 (<strong c...

Watch this Scientific Journal Video about A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s at JoVE.com

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

&#945;-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.

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

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted ...

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