Name: mass spectrometry and luminogenic-based approaches to characterize phase i metabolic competency of in vitro cell cultures
Uploaded: 2017-03-28T14:00:00
Duration: 10 min 44 s
Description: Watch this Scientific Journal Video about Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures at JoVE.com

The authors thank Epithelix Sarl and Biopredic International for providing the airway cells and hepatic cells micrographies and Neil Smith for the figure illustrating the experimental procedure.

<ol>
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A.B. and E.M. were employees of British American Tobacco at the time this manuscript was written. The work described in this manuscript was funded by British American Tobacco, and publication fees for this video-article were paid by British American Tobacco.

Currently, many standardized toxicological test systems use engineered bacteria, cell lines, or embryonic cells that are not representative of the normal human metabolism1. This can lead to inaccurate predictions of the potential toxic activity of a chemical or medicine. Innovative in vitro models, such as 3D cell cultures and organ on a chip, are being developed in an attempt to better replicate the normal morphology and metabolic activity of human tissues21,22,23. Metabolic competency is one criterion that can be used to decipher which models are best suited for toxicological assessment and drug biotransformation studies.
The protocol we have outlined in this paper is designed to measure the activity of CYP enzymes using live cells. It is flexible and can be used for different cell systems in 2D or 3D cultures and offers the option to use either a luminogenic probe- or a mass spectrometry-based approach. Both methods are sensitive enough to detect nanogram quantities of materials and only require a small number of cells grown in a multiwell format. They can also be applied to spheroid or cells grown in complex matrices. The selection of the probe substrate is obviously a critical element of the protocol. The specificity of the assay relies on the probe being selective of the CYP enzyme and another layer of assurance can be obtained by the use of a selective CYP inhibitor. The substrates and inhibitors listed in this paper are just a few examples, but others can be found in the literature.
It is important to consider multiple incubation times when working with a new cell type as a negative CYP activity result could be due to the enzyme being expressed at a low level. The addition of a cell type that can be used as a positive control is advisable to ensure that a negative result is not linked to a technical issue and to help with any troubleshooting. Primary hepatic cells are metabolically competent and can be used as control, for example primary hepatocytes in suspension are very easy to use but their viability is limited in time. Hepatic cell lines are possible alternatives which are relatively easy to use as described in this protocol, but some are better than others in terms of metabolic activity6,21.
Since the assay is non-destructive, unless total protein is quantified, it is possible to follow the CYP activity over time in longitudinal studies20. This is an important point because some cells will have variable CYP activity over time in culture. A negative result might be associated with the lack of confluency, progress with the differentiation or dedifferentiation in vitro. In that case, the approach is semi-quantitative since the data is not normalized by the total amount of protein in each cell culture. It is not always possible or recommended to reuse the tissue after measuring the CYP activity as exposure to probes, inhibitors or inducers can have a toxic effect on the tissue as is the case for TCDD.
Cell fractions such as microsomes could also be used to characterize the CYP activity of a given cell type. Microsome preparations, however, require a lot of cell material, the addition of adequate cofactors and buffer to ensure that the enzymes remain active. Using live cells eliminates the tricky microsome preparation step and working out which buffers and cofactors work best.
As a general recommendation, it is a best practice to further characterize the expression profile of CYPs in cell cultures to verify that it matches the enzymatic activity. This additional level of verification is recommended given that the metabolic probes are not entirely specific to a single CYP and it gives increased confidence that the measured metabolic activity is matched by the corresponding enzyme expression. Since CYPs are membrane proteins they are relatively difficult to prepare for western blots, therefore quantitative RT-PCR has been the preferred approach to profile these enzymes20.
It is important to note that this protocol describes a method to assay CYP enzyme activity that only represents one family of metabolic enzymes, albeit an important one. The flexibility of a mass spectrometry approach allows the development of acquisition methods for other metabolic transformations of probe substrates. However, those have to be developed one at a time and there are currently fewer known specific probes and selective inhibitors for other metabolic enzyme families. This gap should be addressed to allow for a comprehensive assessment of the metabolic competency of in vitro cell systems.

Xenobiotic metabolism is the process through which chemicals foreign to the body are modified by the addition of hydrophilic groups and conjugates to facilitate their excretion1. Typically the metabolism of xenobiotics is a two-step process with Phase I consisting mostly of an oxidation with the addition of one or multiple hydroxyl groups1,2. In Phase II, the hydroxyl groups are used as acceptors for a hydrophilic conjugate such as glucuronide or a sulfate moiety1,2. If an acceptor group is already present on the molecules, the conjugation step can happen independently of Phase I metabolism. Each reaction is performed by a specific group of enzymes, e.g., CYPs (cytochrome P450s), that catalyze the hydroxylation (Figure 1) and dealkylation of chemical substrates3. Conjugation is catalyzed by sulfotransferases, UDP-glucuronosyltransferases (Figure 1), glutathione-S-transferases and N-acetyltransferases4. Each tissue and organ will have a specific metabolic enzyme expression profile, with the liver expressing most of those proteins.
<img alt="Figure 1" src="/files/ftp_upload/55502/55502fig1.jpg" /> 
Figure 1: Example of Phase I and Phase II Metabolism for Coumarin. CYP2A6/CYP2A13 catalyze the 7-hydroxylation of coumarin. 7-hydroxycoumarin is conjugated to a glucuronide moiety by phase II enzymes UGTs, with UGT1A6 and UGT1A9 showing the highest activity.
Understanding the metabolism of xenobiotics is critical in the evaluation of drug safety and for chemical risk assessment for two reasons: the reaction kinetics will determine how long a drug or chemical will remain in the body in an active or inactive form before excretion; and the parent compound can be modified to a more reactive, unstable and toxic species by metabolic enzymes. Such reactions also known as &#34;bioactivation&#34; are mostly driven by phase I CYP enzymes but also on rarer occasions by phase II conjugation5.
Based on this, the ability of an in vitro model to accurately predict the risk associated with a drug or a chemical is highly dependent on the metabolic competency of the cell system. Cell lines derived from diseased tissues or from transformation of normal cells often lose part if not all of the metabolic enzyme profile representative of their tissue of origin6. The maintenance of the normal metabolic enzyme profile appears better in primary cell cultures (at least in short term cultures) and is further improved if the tissue is cultured in a matrix allowing it to retain its 3D structure1. Therefore, the characterization of the metabolic competency of an in vitro cell system is an important preliminary step in guiding the decision regarding which cell model is appropriate to conduct chemical safety evaluations.
In this paper we present a protocol to profile the activity and expression of Phase I CYP enzymes in vitro with examples of their application with a hepatic cell line7 and 3D primary lung cell cultures8. CYP specific substrates, their metabolic products and inhibitor controls are described along with mass spectrometry- and luminogenic-based quantification methods. Since some CYPs are inducible and others are constitutive, examples will also be given for those two scenarios.

<table border="0" cellpadding="0" cellspacing="0" width="697">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Reagent &#38; Cells</td>
			<td style="width:156px;"></td>
			<td style="width:203px;"></td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 &#956;m syringe filter</td>
			<td>Whatman</td>
			<td>UN203NPEORG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well plate</td>
			<td>Corning</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-methylumbelliferone</td>
			<td>Sigma Aldrich</td>
			<td>M1381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-phenyl pentyne</td>
			<td>Sigma Aldrich</td>
			<td>CD5001437</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-hydroxybupropion</td>
			<td>Sigma Aldrich</td>
			<td>H3167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-hydroxychlorzoxazone</td>
			<td>Sigma Aldrich</td>
			<td>UC148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7-ethoxycoumarin</td>
			<td>Sigma Aldrich</td>
			<td>195642</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7-hydroxycoumarin</td>
			<td>Sigma Aldrich</td>
			<td>H24003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">8-methoxypsoralen</td>
			<td>Sigma Aldrich</td>
			<td>M3501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">acetic acid</td>
			<td>Sigma Aldrich</td>
			<td>695092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">acetonitrile</td>
			<td>Fisher</td>
			<td>A/0626/17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#945;-naphthoflavone</td>
			<td>Sigma Aldrich</td>
			<td>N5757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BCA kit</td>
			<td>Thermo Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">b-glucuronidase/arylsulfatase</td>
			<td>Sigma Aldrich</td>
			<td>G0876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bupropion</td>
			<td>Sigma Aldrich</td>
			<td>B102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carbamazepine</td>
			<td>Sigma Aldrich</td>
			<td>C4024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">chlorzoxasone</td>
			<td>Sigma Aldrich</td>
			<td>C4397</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">collagen</td>
			<td>Cell Systems</td>
			<td>5005-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">coumarin</td>
			<td>Sigma Aldrich</td>
			<td>C4261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">disulfiram</td>
			<td>Sigma Aldrich</td>
			<td>86720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fluvoxamine</td>
			<td>Sigma Aldrich</td>
			<td>F2802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutamax (Glutamine supplement)</td>
			<td>Fisher</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HepaRG metabolism supplement</td>
			<td>Merck</td>
			<td>MMAD621</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HepaRG thaw media supplement</td>
			<td>Merck</td>
			<td>MMADD671</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HepaRG</td>
			<td>Merck</td>
			<td>MMHPR116</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luciferin-CEE</td>
			<td>Promega</td>
			<td>V8751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">methanol</td>
			<td>Fisher</td>
			<td>M/4056/17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MucilAir airway cells</td>
			<td>Epithelix</td>
			<td>EP01</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">MucilAir airway cells maintenance media</td>
			<td>Epithelix</td>
			<td>EP04MM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Phenomenex Kinetex 2.6&#956;m, PFP 100A</td>
			<td>Phenomenex</td>
			<td>00B-4477-AN</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TCDD</td>
			<td>Sigma Aldrich</td>
			<td>48599</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;">thioTEPA</td>
			<td>Sigma Aldrich</td>
			<td>T6069</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:276px;">Waters Acquity UPLC HSS T3 1.8&#181;m 2.1 x 50mm</td>
			<td>Waters</td>
			<td>86003538</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Williams&#8217; E media</td>
			<td>Fisher</td>
			<td>17704-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UPLC</td>
			<td>Waters</td>
			<td>Acquity</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QTRAP MS</td>
			<td>Sciex&#160;</td>
			<td>ABI Sciex 4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QTRAP MS software</td>
			<td>Sciex&#160;</td>
			<td>Analyst 1.4.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">luminometer</td>
			<td>Molecular Devices</td>
			<td>SpectraMax M3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">spectrophotometer</td>
			<td>Molecular Devices</td>
			<td>SpectraMax M3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cell counter</td>
			<td>Beckman Coulter</td>
			<td>Vi-Cell XR</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rotary evaporator</td>
			<td>Eppendorf</td>
			<td>Eppendorf-5301</td>
			<td></td>
		</tr>
	</tbody>
</table>

mass spectrometry and luminogenic-based approaches to characterize phase i metabolic competency of in vitro cell cultures

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase solubility and facilitate excretion. On some occasions those structural modifications lead to the formation of new toxic products. In order to reduce animal testing, chemical risk can be assessed using metabolically competent cells. The expression of metabolic enzymes, however, is not stable over time in many in vitro primary culture systems and is often partial or absent in cell lines. Therefore, the study of medicines, additives, and environmental pollutants metabolism in vitro should ideally be conducted in cell systems where metabolic activity has been characterized. We explain here an approach to measure the activity of a class of metabolic enzymes (Human Phase I) in 2D cell lines and primary 3D cultures using chemical probes and their metabolic products quantifiable by UPLC mass spectrometry and luminometry. The method can be implemented to test the metabolic activity in cell lines and primary cells derived from a variety of tissues.

Examples of metabolic activity measured for two pairs of CYPs (CYP1A1/1B1 and CYP2A6/2A13) in the airway cell cultures from three different donors (airway M360, M370, M417) and the hepatic cell line (Hep line) are shown in Figure 620.
CYP activity measured by luminometry 
Figure 6A shows the relative luminescence produced by the CYP1A1/1B1 O-dealkylation activity of Luc-CEE (Luciferin 6&#39;-chloroethyl ether) in airway cells (3 donors) and hepatic cells with and without TCDD induction. CYP1A1/1B1 are highly inducible enzymes and are typically not expressed at a constitutive level in most tissues. As can be seen in Figure 6A, no luminogenic signal is detected when the cells are not induced. When both cell types are pre-incubated with TCDD, a strong CYP1A1/1B1 inducer, a marked luminogenic signal is detected compared to the control (Figure 6A). This indicates that the Luc-CEE probe has been metabolized leading to the release of the luciferin moiety that is quantified subsequently. Overall the activity from the TCDD treated hepatic cell line is lower than for the airway cells, even after total protein normalization. This is not unusual since cell lines tend to be less metabolically active than primary cells. Addition of &#945;-naphthoflavone has a clear inhibitory effect on CYP1A1/1B1 activity in all cell types which further confirms the CYP-dependency of Luc-CEE metabolism.
Quantification of CYP450 metabolites using UPLC-MS 
Figure 6B illustrates the quantification of CYP2A6/2A13 activity in airway cells from 3 donors (Airway M343, M345, M347) and hepatic cells (Hep line) after incubation with coumarin in the presence and absence of the inhibitor 8-methoxypsoralen. The data is normalized for total protein. In the 0 min coumarin incubation control, no 7-hydroxycoumarin is detected (Figure 6B). CYP2A6/2A13 are expressed constitutively in certain tissues and therefore after 16 h incubation with coumarin formation of 7-hydroxycoumarin is detected in the different cell types tested, even without induction. When 8-methoxypsoralen, a CYP2A inhibitor is added (Figure 6B), the formation of 7-hydroxycoumarin is considerably reduced.
It should be noted that in the presence of the CYP inhibitor, it is normal to still detect some residual activity as inhibition is rarely complete and since other CYPs might also be able to recognize the substrate but process it far less efficiently. It can also be observed that the recorded metabolic activity can be different between donors when using primary cells as it is visible for CYP2A6 in airway cells (Figure 6B). This is expected as CYP activity is liable to each donor&#39;s genotype and polymorphisms.
<img alt="Figure 6" src="/files/ftp_upload/55502/55502fig6.jpg" /> 
Figure 6: Activity Assay Result for CYP1A1/1B1 Activity (A) and CYP2A6/2A13 activity (B) in the Hepatic Cell Line and in Three Airway Cell Donors (M360, M370, M417). (A) Luc-CEE dealkylation activity is shown with and without induction of CYP1A1/1B1 by TCDD. &#945;-naphthoflavone was used as CYP1A1/1B1 inhibitor. Activity is expressed as relative luminescence units (RLU) normalized by incubation time in minutes (min) and total protein (mg). (B) CYP2A6/2A13 do not require induction as they are expressed constitutively. Coumarin 7-hydroxylation activity in pmol/min/mg proteins is shown at different time points with and without the inhibitor 8-methoxypsoralen. All results are presented as a mean value of three independent measurements with standard deviations of the mean. This figure was modified from Baxter20.
Supplemental Table 1: UPLC and Mass Spectrometer Settings. <a href="http://cloudflare.jove.com/files/ftp_upload/55502/Supplemental_Table_1.xlsx" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures at JoVE.com

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Metabolic competency of in vitro systems is a key requirement for the biotransformation and disposition of drugs and toxicants. In this protocol, we describe the application of reference metabolic probes to assess phase I metabolism in cell cultures.

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase ...

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