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

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.