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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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation+of+methyl+and+ethyl+nitrosamines+by+cytochrome+P450+2E1+and+2B1.">Oxidation of methyl and ethyl nitrosamines by cytochrome P450 2E1 and 2B1.</a> Biochemistry. 51 (50), 9995-10007 (2012).</li><li>Chowdhury, G., Calcutt, M. W., Guengerich, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation+of+N-nitrosoalkylamines+by+human+cytochrome+P450+2A6:+sequential+oxidation+to+aldehydes+and+carboxylic+acids+and+analysis+of+reaction+steps.">Oxidation of N-nitrosoalkylamines by human cytochrome P450 2A6: sequential oxidation to aldehydes and carboxylic acids and analysis of reaction steps.</a> J Biol Chem. 285 (11), 8031-8044 (2010).</li><li>Carlson, E. S., Upadhyaya, P., Hecht, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+nitrosamide+formation+in+the+cytochrome+P450-mediated+metabolism+of+tobacco-specific+nitrosamines.">Evaluation of nitrosamide formation in the cytochrome P450-mediated metabolism of tobacco-specific nitrosamines.</a> Chem Res Toxicol. 29 (12), 2194-2205 (2016).</li><li>Amin, S., Desai, D., Hecht, S. S., Hoffmann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+tobacco-specific+N-nitrosamines+and+their+metabolites+and+results+of+related+bioassays.">Synthesis of tobacco-specific N-nitrosamines and their metabolites and results of related bioassays.</a> Crit. Rev. Toxicol. 26, 139-147 (1996).</li><li>Clark, A. G., Wong, S. 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. F., 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=Importance+of+purity+evaluation+and+the+potential+of+quantitative+(1)H+NMR+as+a+purity+assay.">Importance of purity evaluation and the potential of quantitative (1)H NMR as a purity assay.</a> J Med Chem. 57 (22), 9220-9231 (2014).</li><li>van der Heeft, E., 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=A+microcapillary+column+switching+HPLC-electrospray+ionization+MS+system+for+the+direct+identification+of+peptides+presented+by+major+histocompatibility+complex+class+I+molecules.">A microcapillary column switching HPLC-electrospray ionization MS system for the direct identification of peptides presented by major histocompatibility complex class I molecules.</a> Anal Chem. 70 (18), 3742-3751 (1998).</li><li>White, E. 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 ...

The overall goal of this methodology is to determine if Cytochrome P450s can processively oxidize n nitrosamines to n nitosamides in vitro. This method benefits the toxicology field by evaluating if nitosamines are directly metabolized to nitrosamides. Because nitrosamides are relatively stable in alkylate DNA, they could play a role in carcinogenesis.The main advantage to this technique is the use of a highly sensitive mass spectrometer that gives detection limits lower than previously achieved for similar in vitro nitrosamine metabolism assays. To begin this procedure, place a 25 milliliter round bottom flask, containing a magnetic stir bar, into an oven. Dry for 16 hours at 140 degrees Celsius.After this, transfer the flask to a fume hood and cool it under a stream of nitrogen gas. Add 31.8 milligrams of norcotinine, five milliliters of acetic anhydride, and one milliliter of acetic acid to the flask, and begin stirring. Transfer the flask to an ice water bath.Add 33.3 milligrams of sodium nitrite. Continue stirring for 2.5 hours, then pour the reaction into 18 milliliters of ice cold water to quench the reaction. Immediately transfer the solution to a separatory funnel containing 18 milliliters of DCM, and extract the aqueous solution.Extract the aqueous layer two additional times, using 9 milliliters of DCM each time. Next dry the pooled organics over 100 milligrams of magnesium sulfate for two minutes. After filtering, use a rotary evaporator to concentrate the solution at 30 degrees Celsius to yield a crude yellow oil.Purify the crude compound using column chromatography as outlined in the text protocol. After this, dissolve the pure compound in one milliliter of deuterated chloroform. Using NMR, confirm the structure and molarity of the solution as outlined in the text protocol.Store this solution in the dark at a temperature between two and eight degrees Celsius until needed. Then confirm the accurate parent mass and determine the product ion masses of the pure NNC solution by direct infusion on a high resolution mass spectrometer, as outlined in the text protocol. To begin, remove the need reagents from a 80 degrees Celsius freezer, and thaw on ice.One thawed, use a 0.5 strength reaction buffer to dilute the Baculosomes in NADPH regeneration system 1-10 and 1-50 respectively, in a one milliliter tube. Next add three microliters of the NADP stock solution to a one milliliter tube containing 97 microliters of a 0.5 strength reaction buffer. For each incubation, prepare a one milliliter tube by adding 40 microliters of a four micromolar NNN solution made with reaction buffer.Add 50 microliters of enzyme solution containing five picomoles of P450 2A6 to each tube. Preincubate in a water bath at 37 degrees for two minutes. Then add 10 microliters of diluted NADP+solution to each tube.Incubate at 37 degrees Celsius for between one and 30 minutes. After this, first add 10 microliters of 3.0 normal zinc sulfate, and then add 10 microliters of 3.0 normal barium hydroxide to quench the reaction. Vortex the solution to form a white precipitate.Next, centrifuge at eight thousand Gs for four minutes. Immediately collect the supernatant, and analyze two microliters by liquid chromatography positive nanoelectrospray ionization high resolution tandem mass spectrometry. In this study, norcotinine is nitrosated to NNC cleanly and in high yield, producing a standard for use in the in vitro experiment.The reaction's success is verified by spectroscopic analysis, including high resolution tandem mass spectrometry, which confirmed that the parent's mass is within five parts per million of the theoretical value. Tandem mass spectrometry fragmentation of NNC is then used to determine the most abundant product ion masses. As can be seen, the two most abundant product ions are 134 and 162, with accurate mass detection out to five decimal places.The synthesized NNC is then utilized as a standard for the in vitro P450 metabolism assay. Under the conditions used, synthetic NNC elutes at approximately 17.5 minutes, with well resolved peaks in both the parent's ion and product ion channels. In accordance with the positive control, NNC formation is seen in the one minute, five minute, and 10 minute incubations.If properly performed, the nitrosamide synthesis inspectroscopic analysis can be done in approximately six hours. Likewise, the in vitro assay with multiple incubations should take no more than eight hours. The implications of this technique extend towards risk assessment for tobacco related cancers, because measurement of nitrosamide related biomarkers could be informative about individual nitrosamine activation.While attempting this procedure, it's important to remember to process enzyme incubations quickly. Nitrosamides hydrolyze under these conditions and should not sit long before mass spec analysis. After watching this video, you should have a good understanding of how to prepare nitrosamide standards, and evaluate if they're formed in vitro by the proper nitrosamine cytochrome P450 system.Don't forget that working with NNN, NNC, or related carcinogens can be hazardous. These compounds should always be handled in a fume hood while wearing a lab coat, safety glasses, and gloves. Additionally, all carcinogenic wastes should be properly stored and labeled.

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Research

JoVE Journal

Cancer Research

9.8K Views.  University of Minnesota. The overall goal of this methodology is to determine if Cytochrome P450s can processively oxidize n nitrosamines to n nitosamides in vitro. This method benefits the toxicology field by evaluating if nitosamines are directly metabolized to nitrosamides. Because nitrosamides are relatively stable in alkylate DNA, they could play a role in carcinogenesis.The main advantage to this technique is the use of a highly sensitive mass spectrometer that gives detection limits lower than previousl...