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><li>Garcia-Canton, C., Minet, E. Metabolic Characterization of Cell Systems Used in In Vitro Toxicology Testing. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGarcia-Canton%2C+C+%22Encyclopedia+of+Drug+Metabolism+and+Interactions%22">Encyclopedia of Drug Metabolism and Interactions</a>. , 1-31 (2016).</li>
<li>Croom, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolism+of+xenobiotics+of+human+environments%5BTitle%5D)+AND+%22Prog.+Mol.+Biol.+Transl.+Sci%22%5BJournal%5D)">Metabolism of xenobiotics of human environments.</a> Prog. Mol. Biol. Transl. Sci. 112, 31-88 (2012).</li>
<li>Lewis, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((57+varieties%3A+the+human+cytochromes+P450%5BTitle%5D)+AND+%22Pharmacogenomics%22%5BJournal%5D)">57 varieties: the human cytochromes P450.</a> Pharmacogenomics. 5, 305-318 (2004).</li>
<li>Jancova, P., Anzenbacher, P., Anzenbacherova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phase+II+drug+metabolizing+enzymes%5BTitle%5D)+AND+%22Biomed.+Pap.+Med.+Fac.+Univ+Palacky%22%5BJournal%5D)">Phase II drug metabolizing enzymes.</a> Biomed. Pap. Med. Fac. Univ Palacky. 154, Olomouc. Czech. Repub. 103-116 (2010).</li>
<li>Dekant, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+biotransformation+and+bioactivation+in+toxicity%5BTitle%5D)+AND+%22EXS%22%5BJournal%5D)">The role of biotransformation and bioactivation in toxicity.</a> EXS. 99, 57-86 (2009).</li>
<li>Donato, M. T., Lahoz, A., Castell, J. V., Gomez-Lechon, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+lines%3A+a+tool+for+in+vitro+drug+metabolism+studies%5BTitle%5D)+AND+%22Curr.+Drug+Metab%22%5BJournal%5D)">Cell lines: a tool for in vitro drug metabolism studies.</a> Curr. Drug Metab. 9, 1-11 (2008).</li>
<li>Guillouzo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+human+hepatoma+HepaRG+cells%3A+a+highly+differentiated+model+for+studies+of+liver+metabolism+and+toxicity+of+xenobiotics%5BTitle%5D)+AND+%22Chem.+Biol.+Interact%22%5BJournal%5D)">The human hepatoma HepaRG cells: a highly differentiated model for studies of liver metabolism and toxicity of xenobiotics.</a> Chem. Biol. Interact. 168, 66-73 (2007).</li>
<li>Huang, S., Wiszniewski, L., Constant, S., Roggen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Potential+of+in+vitro+reconstituted+3D+human+airway+epithelia+%28MucilAir%29+to+assess+respiratory+sensitizers%5BTitle%5D)+AND+%22Toxicol.+In+Vitro%22%5BJournal%5D)">Potential of in vitro reconstituted 3D human airway epithelia (MucilAir) to assess respiratory sensitizers.</a> Toxicol. In Vitro. 27, 1151-1156 (2013).</li>
<li>Sisson, J. H., Stoner, J. A., Ammons, B. A., Wyatt, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((All-digital+image+capture+and+whole-field+analysis+of+ciliary+beat+frequency%5BTitle%5D)+AND+%22J.+Microsc%22%5BJournal%5D)">All-digital image capture and whole-field analysis of ciliary beat frequency.</a> J. Microsc. 211, 103-111 (2003).</li>
<li>Kuehn, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+assessment+of+repeated+exposure+of+organotypic+3D+bronchial+and+nasal+tissue+culture+models+to+whole+cigarette+smoke%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Impact assessment of repeated exposure of organotypic 3D bronchial and nasal tissue culture models to whole cigarette smoke.</a> J. Vis. Exp. , (2015).</li>
<li>Draper, A. J., Madan, A., Parkinson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+coumarin+7-hydroxylase+activity+in+human+liver+microsomes%5BTitle%5D)+AND+%22Arch.+Biochem.+Biophys%22%5BJournal%5D)">Inhibition of coumarin 7-hydroxylase activity in human liver microsomes.</a> Arch. Biochem. Biophys. 341, 47-61 (1997).</li>
<li>Jensen, K. G., Poulsen, H. E., Doehmer, J., Loft, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetics+and+inhibition+by+fluvoxamine+of+phenacetin+O-deethylation+in+V79+cells+expressing+human+CYP1A2%5BTitle%5D)+AND+%22Pharmacol.+Toxicol%22%5BJournal%5D)">Kinetics and inhibition by fluvoxamine of phenacetin O-deethylation in V79 cells expressing human CYP1A2.</a> Pharmacol. Toxicol. 76, 286-288 (1995).</li>
<li>Yamazaki, H., Tanaka, M., Shimada, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+sensitive+high-performance+liquid+chromatographic+assay+for+coumarin+7-hydroxylation+and+7-ethoxycoumarin+O-deethylation+by+human+liver+cytochrome+P450+enzymes%5BTitle%5D)+AND+%22J.+Chromatogr.+B.+Biomed.+Sci.+Appl%22%5BJournal%5D)">Highly sensitive high-performance liquid chromatographic assay for coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation by human liver cytochrome P450 enzymes.</a> J. Chromatogr. B. Biomed. Sci. Appl. 721, 13-19 (1999).</li>
<li>Turpeinen, M., Raunio, H., Pelkonen, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+functional+role+of+CYP2B6+in+human+drug+metabolism%3A+substrates+and+inhibitors+in+vitro%2C+in+vivo+and+in+silico%5BTitle%5D)+AND+%22Curr.+Drug+Metab%22%5BJournal%5D)">The functional role of CYP2B6 in human drug metabolism: substrates and inhibitors in vitro, in vivo and in silico.</a> Curr. Drug Metab. 7, 705-714 (2006).</li>
<li>Kartha, J. S., Yost, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanism-based+inactivation+of+lung-selective+cytochrome+P450+CYP2F+enzymes%5BTitle%5D)+AND+%22Drug+Metab.+Dispos%22%5BJournal%5D)">Mechanism-based inactivation of lung-selective cytochrome P450 CYP2F enzymes.</a> Drug Metab. Dispos. 36, 155-162 (2008).</li>
<li>Sheets, P. L., Yost, G. S., Carlson, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Benzene+metabolism+in+human+lung+cell+lines+BEAS-2B+and+A549+and+cells+overexpressing+CYP2F1%5BTitle%5D)+AND+%22J.+Biochem.+Mol.+Toxicol%22%5BJournal%5D)">Benzene metabolism in human lung cell lines BEAS-2B and A549 and cells overexpressing CYP2F1.</a> J. Biochem. Mol. Toxicol. 18, 92-99 (2004).</li>
<li>Kharasch, E. D., Thummel, K. E., Mhyre, J., Lillibridge, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-dose+disulfiram+inhibition+of+chlorzoxazone+metabolism%3A+a+clinical+probe+for+P450+2E1%5BTitle%5D)+AND+%22Clin.+Pharmacol.+Ther%22%5BJournal%5D)">Single-dose disulfiram inhibition of chlorzoxazone metabolism: a clinical probe for P450 2E1.</a> Clin. Pharmacol. Ther. 53, 643-650 (1993).</li>
<li>Graef, V., Furuya, E., Nishikaze, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hydrolysis+of+steroid+glucuronides+with+beta-glucuronidase+preparations+from+bovine+liver%2C+Helix+pomatia%2C+and+E.+coli%5BTitle%5D)+AND+%22Clin.+Chem%22%5BJournal%5D)">Hydrolysis of steroid glucuronides with beta-glucuronidase preparations from bovine liver, Helix pomatia, and E. coli.</a> Clin. Chem. 23, 532-535 (1977).</li>
<li>Guide to achieving reliable quantitative LC-MS measurements. RSC Analytical Methods Committee. , Available from: 
 <a target="_blank" href="http://www.lgcgroup.com/LGCGroup/media/PDFs/Our%20science/NMI%20landing%20page/Publications%20and%20resources/Guides/AMC_LCMS_Guide.pdf">http://www.lgcgroup.com/LGCGroup/media/PDFs/Our%20science/NMI%20landing%20page/Publications%20and%20resources/Guides/AMC_LCMS_Guide.pdf</a> (2013).</li>
<li>Baxter, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+omics+analyses%2C+and+metabolic+enzyme+activity+assays+demonstrate+maintenance+of+key+mucociliary+characteristics+in+long+term+cultures+of+reconstituted+human+airway+epithelia%5BTitle%5D)+AND+%22Toxicol.+In+Vitro%22%5BJournal%5D)">Targeted omics analyses, and metabolic enzyme activity assays demonstrate maintenance of key mucociliary characteristics in long term cultures of reconstituted human airway epithelia.</a> Toxicol. In Vitro. 29, 864-875 (2015).</li>
<li>Gomez-Lechon, M. J., Tolosa, L., Conde, I., Donato, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Competency+of+different+cell+models+to+predict+human+hepatotoxic+drugs%5BTitle%5D)+AND+%22Expert.+Opin.+Drug+Metab.+Toxicol%22%5BJournal%5D)">Competency of different cell models to predict human hepatotoxic drugs.</a> Expert. Opin. Drug Metab. Toxicol. 10, 1553-1568 (2014).</li>
<li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessment+of+metabolism-dependent+drug+efficacy+and+toxicity+on+a+multilayer+organs-on-a-chip%5BTitle%5D)+AND+%22Integr.+Biol%22%5BJournal%5D)">Assessment of metabolism-dependent drug efficacy and toxicity on a multilayer organs-on-a-chip.</a> Integr. Biol. 8, Camb. 1022-1029 (2016).</li>
<li>Takahashi, Y., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/25947886">Three-dimensional (3D) spheroid cultures improve the metabolic gene expression profiles of HepaRG cells.</a> Biosci. Rep. , (2015).</li>
</ol>

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><tbody><tr><td>&lt;strong&gt;Reagent &amp;amp; Cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2 &amp;mu;m syringe filter</td><td>Whatman</td><td>UN203NPEORG</td><td></td></tr><tr><td>24 well plate</td><td>Corning</td><td>3524</td><td></td></tr><tr><td>4-methylumbelliferone</td><td>Sigma Aldrich</td><td>M1381</td><td></td></tr><tr><td>5-phenyl pentyne</td><td>Sigma Aldrich</td><td>CD5001437</td><td></td></tr><tr><td>6-hydroxybupropion</td><td>Sigma Aldrich</td><td>H3167</td><td></td></tr><tr><td>6-hydroxychlorzoxazone</td><td>Sigma Aldrich</td><td>UC148</td><td></td></tr><tr><td>7-ethoxycoumarin</td><td>Sigma Aldrich</td><td>195642</td><td></td></tr><tr><td>7-hydroxycoumarin</td><td>Sigma Aldrich</td><td>H24003</td><td></td></tr><tr><td>8-methoxypsoralen</td><td>Sigma Aldrich</td><td>M3501</td><td></td></tr><tr><td>acetic acid</td><td>Sigma Aldrich</td><td>695092</td><td></td></tr><tr><td>acetonitrile</td><td>Fisher</td><td>A/0626/17</td><td></td></tr><tr><td>&amp;alpha;-naphthoflavone</td><td>Sigma Aldrich</td><td>N5757</td><td></td></tr><tr><td>BCA kit</td><td>Thermo Scientific</td><td>23227</td><td></td></tr><tr><td>b-glucuronidase/arylsulfatase</td><td>Sigma Aldrich</td><td>G0876</td><td></td></tr><tr><td>Bupropion</td><td>Sigma Aldrich</td><td>B102</td><td></td></tr><tr><td>Carbamazepine</td><td>Sigma Aldrich</td><td>C4024</td><td></td></tr><tr><td>chlorzoxasone</td><td>Sigma Aldrich</td><td>C4397</td><td></td></tr><tr><td>collagen</td><td>Cell Systems</td><td>5005-B</td><td></td></tr><tr><td>coumarin</td><td>Sigma Aldrich</td><td>C4261</td><td></td></tr><tr><td>disulfiram</td><td>Sigma Aldrich</td><td>86720</td><td></td></tr><tr><td>fluvoxamine</td><td>Sigma Aldrich</td><td>F2802</td><td></td></tr><tr><td>Glutamax (Glutamine supplement)</td><td>Fisher</td><td>35050061</td><td></td></tr><tr><td>HepaRG metabolism supplement</td><td>Merck</td><td>MMAD621</td><td></td></tr><tr><td>HepaRG thaw media supplement</td><td>Merck</td><td>MMADD671</td><td></td></tr><tr><td>HepaRG</td><td>Merck</td><td>MMHPR116</td><td></td></tr><tr><td>Luciferin-CEE</td><td>Promega</td><td>V8751</td><td></td></tr><tr><td>methanol</td><td>Fisher</td><td>M/4056/17</td><td></td></tr><tr><td>MucilAir airway cells</td><td>Epithelix</td><td>EP01</td><td></td></tr><tr><td>MucilAir airway cells maintenance media</td><td>Epithelix</td><td>EP04MM</td><td></td></tr><tr><td>Phenomenex Kinetex 2.6 &amp;mu;m, PFP 100A</td><td>Phenomenex</td><td>00B-4477-AN</td><td></td></tr><tr><td>TCDD</td><td>Sigma Aldrich</td><td>48599</td><td></td></tr><tr><td>thioTEPA</td><td>Sigma Aldrich</td><td>T6069</td><td></td></tr><tr><td>Waters Acquity UPLC HSS T3 1.8 &amp;micro;m 2.1 x 50 mm</td><td>Waters</td><td>86003538</td><td></td></tr><tr><td>Williams&amp;rsquo; E media</td><td>Fisher</td><td>17704-024</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>UPLC</td><td>Waters</td><td>Acquity</td><td></td></tr><tr><td>QTRAP MS</td><td>Sciex&amp;nbsp;</td><td>ABI Sciex 4000</td><td></td></tr><tr><td>QTRAP MS software</td><td>Sciex&amp;nbsp;</td><td>Analyst 1.4.2</td><td></td></tr><tr><td>luminometer</td><td>Molecular Devices</td><td>SpectraMax M3</td><td></td></tr><tr><td>spectrophotometer</td><td>Molecular Devices</td><td>SpectraMax M3</td><td></td></tr><tr><td>cell counter</td><td>Beckman Coulter</td><td>Vi-Cell XR</td><td></td></tr><tr><td>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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JoVE Journal

Biochemistry

9.8K Views.  British American Tobacco.  Metabole competentie van in vitro systemen is een belangrijke voorwaarde voor de biotransformatie en dispositie van drugs en toxische stoffen. In dit protocol beschrijven we de toepassing van referentie- metabole probes fase I metabolisme in celkweken beoordelen. 

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

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

Immunology and Infection

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

The purpose of this article is to describe a protocol for extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis, particularly, capillary electrophoresis-mass spectrometry.

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but ...

Cancer Research

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

Bioengineering

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with 13C-aniline, while compounds in the sample are derivatized with 12C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. The single-probe, a microscale sampling and ionization device, can be coupled with a mass spectrometer for on-line, rapid SCMS analysis of cellular constituents under ambient conditions. Previously, the single-probe SCMS technique was primarily used to measure cells immobilized onto a substrate, limiting the types of cells for studies. In the current study, the single-probe SCMS technology has been integrated with a cell manipulation system, typically used for in vitro fertilization. This integrated cell manipulation and analysis platform uses a cell-selection probe to capture identified individual floating cells and transfer the cells to the single-probe tip for microscale lysis, followed by immediate mass spectrometry analysis. This capture and transfer process removes the cells from the surrounding solution prior to analysis, minimizing the introduction of matrix molecules in the mass spectrometry analysis. This integrated setup is capable of SCMS analysis of targeted patient-isolated cells present in body fluids samples (e.g., urine, blood, saliva, etc.), allowing for potential applications of SCMS analysis to human medicine and disease biology.

An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. ...

Chemistry

Stable Isotope Tracing & LC-MS Analysis to Measure DHODH and SDH Activities

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

Author Spotlight: Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the cell&#39;s survival requirements. Alternatively, carrying out DBTL cycles in cell-free environments can accelerate this process and alleviate concerns with host compatibility. A promising approach to cell-free metabolic engineering (CFME) leverages metabolically active crude cell extracts as platforms for biomanufacturing and for rapidly discovering and prototyping modified proteins and pathways. Realizing these capabilities and optimizing CFME performance requires methods to characterize the metabolome of lysate-based cell-free platforms. That is, analytical tools are necessary for monitoring improvements in targeted metabolite conversions and in elucidating alterations to metabolite flux when manipulating lysate metabolism. Here, metabolite analyses using high-performance liquid chromatography (HPLC) coupled with either optical or mass spectrometric detection were applied to characterize metabolite production and flux in E. coli S30 lysates. Specifically, this report describes the preparation of samples from CFME lysates for HPLC analyses using refractive index detection (RID) to quantify the generation of central metabolic intermediates and by-products in the conversion of low-cost substrates (i.e., glucose) to various high-value products. The analysis of metabolite conversion in CFME reactions fed with 13C-labeled glucose through reversed-phase liquid chromatography coupled to tandem mass spectrometry (MS/MS), a powerful tool for characterizing specific metabolite yields and lysate metabolic flux from starting materials, is also presented. Altogether, applying these analytical methods to CFME lysate metabolism enables the advancement of these systems as alternative platforms for executing faster or novel metabolic engineering tasks.

The protocols describe high-performance liquid chromatography methods coupled to refractive index or mass spectrometric detection for studying metabolic reactions in complex lysate-based cell-free systems.

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

Neuroscience

Comprehensive Protocol for Cell Identification and Metabolic Analysis Using OPTIR-FISH

Optical Photothermal Infrared-Fluorescence In Situ Hybridization (OPTIR-FISH)

Understanding the metabolic activities of individual cells within complex communities is critical for unraveling their role in human disease. Here, we present a comprehensive protocol for simultaneous cell identification and metabolic analysis with the OPTIR-FISH platform by combining rRNA-tagged FISH probes and isotope-labeled substrates. Fluorescence imaging provides cell identification by the specific binding of rRNA-tagged FISH probes, while OPTIR imaging provides metabolic activities within single cells by isotope-induced red shift on OPTIR spectra. Using bacteria cultured with 13C-glucose as a test bed, the protocol outlines microbial culture with isotopic labeling, fluorescence in situ hybridization (FISH), sample preparation, optimization of the OPTIR-FISH imaging setup, and data acquisition. We also demonstrate how to perform image analysis and interpret spectral data at the single-cell level with high throughput. This protocol&#39;s standardized and detailed nature will greatly facilitate its adoption by researchers from diverse backgrounds and disciplines within the broad single-cell metabolism research community.

Author Spotlight: Integrated OPTIR-FISH for Single-Cell Metabolic and Identity Analysis in Complex Environments

Here, we present a protocol using optical photothermal infrared-fluorescence in situ hybridization (OPTIR-FISH), also known as mid-infrared photothermal-FISH (MIP-FISH), to identify individual cells and understand their metabolism. This methodology can be applied broadly for diverse applications, including mapping cellular metabolism with single-cell resolution.

Understanding the metabolic activities of individual cells within complex communities is critical for unraveling their role in human disease. Here, we ...

Low-Input Metabolic Studies in Mice Cell Types

Targeted Metabolomics on Rare Primary Cells

Cellular function critically depends on metabolism, and the function of the underlying metabolic networks can be studied by measuring small molecule intermediates. However, obtaining accurate and reliable measurements of cellular metabolism, particularly in rare cell types like hematopoietic stem cells, has traditionally required pooling cells from multiple animals. A protocol now enables researchers to measure metabolites in rare cell types using only one mouse per sample while generating multiple replicates for more abundant cell types. This reduces the number of animals that are required for a given project. The protocol presented here involves several key differences over traditional metabolomics protocols, such as using 5 g/L NaCl as a sheath fluid, sorting directly into acetonitrile, and utilizing targeted quantification with rigorous use of internal standards, allowing for more accurate and comprehensive measurements of cellular metabolism. Despite the time required for the isolation of single cells, fluorescent staining, and sorting, the protocol can preserve differences among cell types and drug treatments to a large extent.

Author Spotlight: Advancing Metabolomics Analysis of Rare Hematopoietic Stem Cells 

Here, we present a protocol to accurately and reliably measure metabolites in rare cell types. Technical improvements, including a modified sheath fluid for cell sorting and the generation of relevant blank samples, enable a comprehensive quantification of metabolites with an input of only 5000 cells per sample.

Cellular function critically depends on metabolism, and the function of the underlying metabolic networks can be studied by measuring small molecule ...

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

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