The goal of this procedure is to determine the activity of cytochrome P450s, or CYPs, in cultured cells. CYPs are key enzymes involved in the metabolism of medicines and toxicants and their expression is not often retained in vitro. Normal expression of CYP enzymes is often lost when cells are cultured in vitro.
Therefore, many in vitro models are potentially limiting relevance to understanding the metabolism of medicines and toxicants. The main advantage of this method is its simplicity when assessing CYP activity in cultured cells. Using probe substrates and inhibitors, different quantification platforms are presented, including mass spectrometry.
To begin, obtain cryogenic vials with cells as described in the text protocol. The protocol provided uses a hepatic cell line as an example. Whilst working under sterile conditions, carefully open the cryogenic vial to release any excess pressure, then pipette the contents of the cryogenic vial into a 10 milliliter tube filled with nine milliliters of pre-warmed Williams'E medium at 37 degrees Celsius.
Centrifuge the solution for 10 minutes at 400 times g at room temperature. After discarding the supernatant, re-suspend the cells in five milliliters of proprietary thaw medium, pre-warmed to 37 degrees Celsius and supplemented with glutamine. After counting the viable cells as described in the text protocol, seed the cells in a 24 well collagen-coated plate at a density of 60, 000 viable cells per well and a final volume of 0.5 milliliters of thaw medium.
After a few days in culture, the cells form a subconfluent trabecular structure. The optimum metabolic activity is typically observed between days seven and 10, and is lowest at day four. To prepare the differentiated airway epithelium cultures, use commercially available fully differentiated primary airway epithelium that have been grown at the air-liquid interface on an insert.
Using sterile tweezers, gently dislodge the cell insert from the agarose matrix and transfer it to the new plate pre-loaded with 700 microliters of proprietary culture medium. Assess the integrity of the airway tissue using methods that are referenced in the text protocol, such as transepithelial resistance or cilia beat frequency. Prior to the experiment, prepare two series of cell inserts for the airway cells and wells for the hepatic cells with a minimum of three replicates.
Use one series for the inhibition experiment and the other for the metabolic probe only. Prepare a third series of cells as a media blank control. On the day of the experiment, replace the cell culture medium with 450 microliters of fresh medium.
Add 50 microliters of medium containing the 10x CYP inhibitor to the series of cells prepared for the inhibition experiment and pre-incubate for 30 minutes. Next, replace the medium in the inhibition series with 450 microliters of fresh medium, and add 50 microliters of medium containing both 10x CYP inhibitor and the 10x probe substrate. Add 50 microliters of medium with the 10x probe substrate to the other cells, then add an equivalent amount of vehicle diluted in medium to the blank cell controls.
After incubating the cells for zero to five minutes for the time point one, zero background control, collect the medium in separate labeled tubes and freeze the medium for future processing. Then incubate the time point two cells for 16 hours in the cell incubator. At the end of the incubation period, collect the medium using the same method for quantification of the metabolic product.
Transfer 250 microliters of incubation medium to a 1.5 milliliter microtube, and add 4-methylumbelliferone internal standard stock solution to reach a final concentration of 100 nanomolar. Adjust the pH of the medium to between 4.5 and 5.0 by adding one molar HCl, one microliter at a time. Verify the pH by pipetting two to 10 microliters of medium onto a pH strip, then add 150 units of betaglucuronidase arylsulfatase from Helix pamatia.
After incubating for one hour at 37 degrees Celsius, stop the reaction by adding 250 microliters of cold methanol. Evaporate the solvent using a vacuum centrifuge at 45 degrees Celsius. Reconstitute the samples with 500 microliters of a 30%acetonitrile to water solution.
To perform mass spectrometry based quantification, add 250 microliters of each serial dilution standard to an amber ultra performance liquid chromatography glass vial for injection in the UPLC-MS auto sampler. Also add the test samples to amber UPLC vials and place them in the UPLC auto sampler. After randomizing all the vials, run the UPLC-MS using the settings detailed in the text protocol depending on the specific probe to be quantified.
Use the Quantitate, Build Quantitation Method tool in the mass spectrometer quantification tools to generate the calibration curve from the acquired synthetic standard and internal standard, then use the Quantitation Wizard to select the sample batch and samples to quantify and execute the quantitation method. To induce CYP1A1, CYP1B1, use the plate layout found in the text protocol. Incubate the cells designated for induction with a final concentration of 10 nanomolar TCDD in medium for a period of 72 hours.
After this period, remove the medium, rinse the wells with PBS three times, and add some fresh medium. Prior to incubating the cells with the luminogenic reporter probe, pre-incubate the designated subset of induced cells for 30 minutes with the CYP1A1, CYP1B1 inhibitor in medium. Add the luminogenic propbe, luciferin 6'chloroethyl ether at a final concentration of 50 micromolar to the non-induced, induced and induced plus inhibitor cells.
Incubate for four hours and then transfer 25 microliters of culture medium to a white, opaque 96 well plate and add 25 microliters of luciferin detection reagent supplied by the manufacturer. After incubating for 20 minutes at room temperature, read the plate immediately using a luminometer. The relative luminescence produced by the dealkylation of luciferin 6'chloroethyl ether, which is catalyzed by CYP1A1, CYP1B1, are presented here.
The example shows no enzyme activity in the absence of TCDD induction. Following TCDD induction, CYP1A1, CYP1B1 activity is observed in both the hepatic and respiratory cell types. Finally, addition of the CYP1A1, CYP1B1 inhibitor alpha-naphthoflavone reduces or eliminates the CYP activity.
To assess CYP2A6, 2A13 activity in hepatic and airway cell types, the amount of 7-hydroxycoumarin produced through hydroxylation of coumarin by the enzyme was measured using UPLC LC-MS. At zero minutes incubation with coumarin, only background is detected. After 16 hours, hydroxylation of coumarin is observed with the formation of 7-hydroxycoumarin in the two cell types.
Finally, the addition of the CYP2A, 2A13 inhibitor prevents the formation of 7-hydroxycoumarin. Once mastered, the probe incubation and quantification parts of this technique can be done in a few hours to up to a day in most cases. After watching this video, you should have a good understanding of how to perform various phase one metabolism assays using different cell types, confirming the specificity of the enzyme using inhibition and/or induction as required.
While attempting this procedure, it's important to remember that different cell types might require longer incubation times, and it is essential to include a cell type that can be used as a positive control. Don't forget that working with TCDD can be extremely hazardous, and precautions, such as full personal protective equipment should always be taken while performing this procedure. The implications of this technique extend towards using suitable in vitro models for pre-clinical evaluation of novel medicines and the risk assessments of chemicals and ingredients used in consumer products.
