TD-GC-MS Analysis of Volatile Metabolites of Human LungCancer and Normal Cells In vitro
Wojciech Filipiak1,3, Andreas Sponring1,3, Anna Filipiak1,3, Clemens Ager1,3, Jochen Schubert3,4,Wolfram Miekisch3,4, Anton Amann1,3, and Jakob Troppmair1,2
Analysis of exhaled breath is a noninvasive method fordiagnosis and therapeutic monitoring (1-3). Paradigmaticexamples are the 13C-urea breath test for detection ofHelicobacter pylori (4, 5) and the hydrogen-based breath
ffiliations: 1Department of Operative Medicine and 2Daniel-Research Laboratory, Department of Visceral, Transplantic Surgery, Center of Operative Medicine, Innsbruck MedicalInnsbruck, Austria; 3Breath Research Institute of the Austrianf Sciences, Dornbirn, Austria; and 4University of Rostock,t of Anesthesiology and Intensive Care, Rostock, Germany
ontributions: The original plan of the cell culture workpackageroject BAMOD was devised and written by J. Schubert, W.. Amann, and J. Troppmair. W. Filipiak developed the protocol-MS analyses of volatile compounds in headspace of cellonditions of sample collection, thermal desorption, gasraphy temperature program, and mass spectrometry settings)., W. Filipiak performed the gas chromatographic analysis of allerformed the calibrations, and wrote a draft of the manuscript.g contributed to cell culture sampling system development,the cell culture experiments and wrote the draft of the. A. Filipiak performed the chromatographic data analysis. C.he data analysis. J. Schubert and W. Miekisch developedsition of sorption traps and chose the chromatographicAmann and J. Troppmair designed the study, supervised thets, discussed the results and continuous improvement ofents using different analytic techniques, and finalized the. All authors read and approved the manuscript.
ding Author: Anton Amann and Jakob Troppmair, Innsbruckiversity, Innrain 66, Innsbruck 6020, Austria. Phone: 43-512-Fax: 43-512-50424624. E-mail: [email protected]@i-med.ac.at
8/1055-9965.EPI-09-0162
erican Association for Cancer Research.
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test for carbohydrate malabsorption (6). Promising inves-tigations included critically ill persons (7, 8), patients suf-fering from renal and liver diseases (9-13), and cancerpatients (14-21). Typical compounds in exhaled breathcomprised hydrocarbons, ketones, aldehydes, alcohols,amides, sulfides, and ethers (21).Because the field of breath analysis is relatively new, and
the advances in analytic technology occur so fast, manycompounds in exhaled breath have been detected; com-poundswhose biochemical origin has not yet been studied.In addition, little is known about their relationship to cel-lular processes such as malignant transformation. Investi-gations of exhaled breath from cancer patients showed thatconcentrations of specific compounds may be increased ordecreased in comparison with healthy age-matched con-trols. This also applies to compounds in the headspace ofcell cultures. Tumors are complex systems with a high de-gree of heterogeneity. Apart from the transformed cells,nontumorous components may also contribute to volatileorganic compounds (VOC) present in the exhaled air of alung cancer patient. Such potential sources of VOCs,whichhave not been studied here, are the activated immune sys-tem (22-25) and, possibly, microorganisms (26-28). The aimof the present work was to test for the existence of cancer-derivedVOCs through the analysis of established cell lines
Sponring A, Filipiak W, Mikoviny T, et al. Release of volatile organicompounds (VOCs) from the lung cancer cell line NCI-H1666 in vitro.
(29-32). In previous experiments, we investigated threelung cancer cell lines, NCI-H2087 (32), CALU-1 (31), andNCI-H1666.5 In NCI-H2087 cells, the release of the alcohol2-ethyl-1-hexanol and the branched alkane 2-methylpen-tane was observed as well as a decline of acetaldehyde,2-methylpropanal, 3-methylbutanal, 2-methylbutanal,and n-butyl acetate (32). The cell line CALU-1 showed asignificant release of branched hydrocarbons such as2,3,3-trimethylpentane, 2,3,5-trimethylhexane, and 2,4-di-methylheptane and 4-methyloctane, whereas decreasedconcentrations were found for acetaldehyde, 3-methyl-butanal, n-butyl acetate, acetonitrile, acrolein, methacro-lein, 2-methylpropanal, 2-butanone, methyl tert-butylether, ethyl tert-butyl ether, and hexanal (31). However,no significantly increased release of VOCs could beshown for NCI-H1666 cells,5 whereas a decrease inmethacrolein, 3-methylbutanal, hexanal, and n-butyl ac-etate was observed. Thus, in our studies, compoundsespecially belonging to the class of branched hydrocar-bons were released from lung cancer cells in vitro,whereas, in particular, aldehydes and n-butyl acetatedecreased in concentration.In the work presented here, we studied an additional
lung cancer cell line, A549, to obtain a better definedspectrum of potential tumor cell–derived VOCs, as wellas two nontransformed cell lines to filter out substancespotentially restricted to transformed cells.
Materials and Methods
Cell CultureA549 cells, which carry a mutated K-Ras but a
wild-type B-Raf gene, have been obtained from AmericanType Culture Collection. They have been isolated origi-nally from a lung carcinoma of a 58-y-old man andshowed epithelial morphology and grew adherent(33-36). Human fibroblasts (hFB) derived from the dermisare a generous gift of Prof. Gabriele Werner-Felmayer,Section of Biological Chemistry, Biocenter, InnsbruckMedical University, Innsbruck, Austria. A549 and hFBcells were grown in DMEM high-glucose culture mediumcontaining sodium pyruvate (110 mg/L) supplementedwith 10% FCS, penicillin (100,000 units/L), streptomycin(100 mg/L), and L-glutamine (293 mg/L).Human bronchial epithelial cells (HBEpC) are primary
cells (PromoCell GmbH) isolated from the mucosa of themain bronchi of a 42-y-old male Caucasian. The cells werecultivated inAirway Epithelial Cell GrowthMedium (Pro-moCell GmbH) supplemented with the Airway EpithelialCell Growth Medium Supplement Pack (PromoCellGmbH) according to the manufacturer's instructions.For all experiments, cells were cultivated under stan-
dard conditions at 37°C in a humidified atmosphere with92.5% air/7.5% CO2. For VOC measurements, 25, 75, or100 million A549 cells and 50 million HBEpC or hFB cells,respectively, were inoculated in 100 mL phenol red–freeDMEM high-glucose medium (supplements: 5% FCS,100,000 units/L penicillin, 100 mg/L streptomycin, 293
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mg/L L-glutamine, and 110 mg/L sodium pyruvate) orstandard tissue culture medium (HBEpC cells). The con-centration of FCS in DMEM during the experiment waslowered to 5% to reduce the high background of VOCs inthe analyzed headspace. Culture vessels were thenflushed with clean, synthetic air from a gas cylinder (de-fined gas mixture, Linde) containing 5% CO2 to reducebackground contamination. The rinsing was done for 10min at a flow of 100mL/min. Subsequently the fermenterswere sealed for 21 h. At the end of the incubation time, 200mL of air from the headspace was sampled and analyzedby gas chromatography mass spectrometry (GC-MS).
SamplingGlass tubes (Gerstel) filled with the following sorbents
were used as traps for sample collection with simulta-neous preconcentration: 25 mg Tenax TA (60/80 mesh),35 mg Carboxen 569 (20/45 mesh), and 250 mg Carboxen1000 (80/100 mesh; each from Supelco). Sorbents wereseparated by glass wool. To decrease the relative humid-ity, the gaseous samples were diluted 1:6 (5 mL/min sam-ple:25 mL/min air) with dry, additionally purified airtaken from a gas cylinder (Linde). This procedure pre-vents the excessive adsorption of water on carbon molec-ular sieves and thereby avoids problems during samplepreconcentration, cryofocusing during desorption, andfinally chromatographic separation. The volume of col-lected sample originating from the fermenter was 200mLwith a total flow through sorption trap of 30 mL/min.
Thermal DesorptionThe sampled analytes were released from sorbents by
thermal desorption in a TDS3 unit equipped with aTDSA2 auto sampler (both from Gerstel). The flow rateof carrier gas through the sorption trap during desorp-tion was 90 mL/min. The initial temperature of 30°Cwas increased to 300°C with a heating rate of 100°C/min (held for 10 min). Liquid nitrogen was used for cryo-focusing the desorbed analytes at −90°C. For subsequentsample injection into the capillary column, the CIS-4 in-jector, which contained a glass liner filled with CarbotrapB (Gerstel), was heated at a rate of 12°C/s up to 320°C(than hold 2 min in splitless mode).
with gass chromatography mass spectrometry) were doneon a 6890N gas chromatograph equipped with a mass se-lective detector 5973N (both from Agilent Technologies)with sample injection by means of thermal desorption (de-scribed in the previous sections). The PoraBond Q capillarycolumn25m×0.32mm×5μm(Varian)was used.The oventemperature programwas as follows: initial 50°C held for 5min, then ramped 5°C/min up to 140°C, held for 5 min,again ramped 5°C/min to 280°C, and held for 4 min. Theconstant flow rate of helium carrier gaswas 2mL/min. TheMS analyses were done in a full scan mode (TIC mode),with a scan range of 20 to 200 amu. Ionization of the sepa-rated compounds was done by electron impact ionization
Cancer Epidemiol Biomarkers Prev; 19(1) January 2010 183
NOTE: CAS numbers (Chemical Abstracts Service), correlation coefficients (R2), and LODs expressed in concentration unit [ppbv]are presented. Average concentrations (ppbv) are given with SDs. The ratio of the average concentrations of the target analytecompared with medium control and the p values of Kruskal-Wallis tests have been calculated for each cell density.
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at 70 eV. The chromatographic data was acquired using theAgilent Chemstation Software (GC-MSData Analysis fromAgilent). The mass spectrum library NIST 2005 wasapplied for the identification of detected compounds.
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Reagents and Standards2,4-Dimethyl-1-heptene, 2,3,5-trimethylhexane and
2,3,3-trimethylpentane were purchased from Chem-SampCo, and 2-pentanone was from Acros Organics.
Table 1. Quantification of VOCs released or taken up (consumed or degraded) by A549 cancer cells (Cont'd)
Volatile Organic Compounds Released by A549 Cells
All other compounds were purchased from SigmaAldrich.
CalibrationFor the quantification of compounds detected in the
headspace of cells and of the medium, an external stan-dard calibration was done. The preparation of gaseous
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standards was done by evaporating liquid substancesin glass bulbs. Each bulb (Supelco) was cleaned withmethanol (Sigma-Aldrich), dried at 85°C for at least20 h, purged with clean nitrogen for minimally 20min, and subsequently evacuated using a vacuumpump (Vacuubrand) for 30 min. Liquid standards(1-3 μL, according to the desired concentration) were
Cancer Epidemiol Biomarkers Prev; 19(1) January 2010 185
injected through a septum by using a GC syringe. Afterthe evaporation of standards, the glass bulb was filledwith nitrogen of purity 6.0 (i.e., 99.9999%, Linde) toequalize the pressure to ambient pressure. Then, the ap-propriate volume (μL) of vapor mixture was transferredby a gas tight syringe (Hamilton) into Tedlar bags (SKC232 Series), which were previously filled with 1.5 litersof nitrogen (99,9999%, additionally purified by means ofcarbon molecular sieves Carboxen 1000).
Statistical AnalysesPutative statistical significance was calculated by the
Kruskal-Wallis test, which is a test to compare samplesfrom two or more groups of independent observations(37). It is a one-way ANOVA and does not assumea normal population, unlike the analogous one-wayANOVA. The Kruskal-Wallis test is a nonparametricversion of the classic one-way ANOVA, and an exten-sion of the Wilcoxon rank-sum test to more than twogroups (37). Additionally, results are presented as meanvalues with SDs.
Results
Our study with CALU-1 cells had shown that onlylonger incubation times (18 hours) allowed for the re-producible detection of significant differences in VOCconcentrations (31). Here, we consistently kept the incu-bation time at 21 hours and observed that after thistime, the average viability was 97.7 ± 1.1% for 25million, 95.3 ± 2.9% for 75 million, and 96.4 ± 6.2%for 100 million A549 cells. Thus, cell culture conditionsdid not cause substantial cell death, which ensured thatthe release of potential VOCs was mostly due to livingcells. Under the same conditions, the average viabilitywas 95.4 ± 2.29% for hFB and 77.9 ± 9.90 for HBEpCcells, which proved more fragile under the experimentalsetting used. The considerable cell death observed in thecase of the HBEpC cells may additionally contribute todifferences in VOC profiles in ways, which will beaddressed in future studies.
Identification and Quantification of VOCs Releasedby Cells In vitroAmong all compounds detected, 132 compounds were
identified not only by spectral library match using theNIST 2005 library but also by determination of their re-tention time based on calibration mixtures of the respec-tive pure standards. The peaks, for which properidentification was not possible (too low library matchand no confirmation by retention time), are not dis-cussed. Generally, the applied TD-GC-MS method ischaracterized by good linearity (even for the lowest con-centrations detected) with correlation coefficients R2 be-ing predominantly higher than 0.99 in calibrationmeasurements. The limits of detection (LOD) for almostall compounds of interest were at the pptv level, beinglowest for hydrocarbons such as 2,4-dimethylhexane
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(0.044 ppbv) or 3-methylheptane (0.048 ppbv). The ap-plied method was also very sensitive for polar analytessuch as propyl acetate (0.078 ppbv), methyl acetate(0.106 ppbv), or 2-methylbutanal (0.134 ppbv). The com-pounds with LOD at the single ppbv level were alcohols,such as 2-ethyl-1-hexanol (9.885 ppbv), ethanol (2.723ppbv), or acetaldehyde (1.517 ppbv). The use of Tedlarbags for the preparation of standard mixtures for TD-GC-MS calibration could be the reason of relatively highLODs (single ppbv level), especially for alcohols that arepartly absorbed in Tedlar material. It should also be not-ed that the selected ion-monitoring mode, which im-proves the sensitivity of MS analyses, was not applied.Instead, full scan mode (Total Ion Chromatogram, TICmode) was chosen to be adequate for the correct identi-fication of a wide range of VOCs detected in the samples.Thus, the measured low LOD with simultaneous low er-rors (expressed by correlation coefficients) testify verygood precision and sensitivity of the applied TD-GC-MS method.
TD-GC-MS Analyses of VOCs in the Headspace ofA549 CellsOur experiments with A549 cells included the analyses
of medium controls (n = 4) and 25 (n = 5), 75 (n = 4), or100 million cells (n = 5). Eight compounds were found tobe increased and seven compounds to be decreased in theheadspace of A549 cells compared with medium control(Table 1). The concentrations of methyl tert-butyl etherand 2-pentanone were found to be increased in allA549 cancer cell samples (Fig. 1). Besides that, the unsat-urated branched hydrocarbons 2-methyl-1-pentene and2,4-dimethyl-1-heptene were found to be significantly el-evated in the headspace of 75 and 100 million A549 cells.No significant differences to medium controls werefound for these two compounds in experiments with 25million cells although concentrations were increased.Furthermore, isobutene and octane showed significantlyincreased concentrations in experiments with 100 millioncells (P = 0.01 and 0.03, respectively) but not with lowercell amounts. In general, relatively low concentrations ofVOCs released by cells and high background levels orig-inating from medium controls resulted in big SDs. There-fore, a considerable amount of cells is required to detectstatistically significant differences in the level of VOCs re-leased by A549 cancer cells and other tested cell lines.Moreover, ethyl tert-butyl ether, acetone, and ethanolwere present at significantly higher concentrations inthe headspace of 25 million and 100 million cancer cellscompared with medium controls. No statistically signifi-cant difference in the amounts of these three VOCs com-pared with medium controls was found in experimentswith 75 million A549 cells (Fig. 1).Among the decreased compounds, the aldehydes 2-
ethylacrolein and 2-methyl-2-butenal were found exclu-sively in the headspace of medium controls and not inthe headspace of cell samples. 2-Methylpropanal wasnot detected in measurements with 75 million cells
(n = 4) but occasionally in measurements of 25 million (2of 5) and 100 million cells (1 of 5; see Table 1). Similarly,pyrrole was found in several but not all measurements.For a few other significantly decreased compounds, thelevels detected were below their LOD (pyrrole andmethacrolein). Only n-butyl acetate and 3-methylbutanalwere present in all samples at concentrations above theirLODs and at significantly lower concentrations in theheadspace of cancer cells (Fig. 1). n-Butyl acetate showedp values below 0.05 in all experiments and, in the case of100 million cells, a p value of 0.01. More detailed informa-tion can be found in Table 1.
TD-GC-MS Analyses of VOCs in the Headspace ofHBEpC and hFB CellsIn the case of the HBEpC, four independent measure-
ments were done for medium controls (n = 4) and threefor 50 million HBEpC cells (n = 3). The concentrations of10 compounds were increased and of 8 compounds de-creased in the headspace of HBEpC cells compared withmedium control (Table 2). As already observed in A549cells, acetone, ethyl tert-butyl ether, 2-pentanone, and
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2,4-dimethyl-1-heptene were released by HBEpC cellsin significant amounts (Fig. 2). The remaining six VOCswith increased concentrations in the headspace ofHBEpC included the three branched hydrocarbons2,3,3-trimethylpentane, 4-methylheptane, and 3-methyl-heptane. The concentration of 2,3,3-trimethylpentanewas below the LOD in the headspace of medium controlsand the other two were not detected in the controls at all.In addition, the alcohol 2-methyl-2-propanol and the es-ters methyl acetate and n-propyl acetate (not detected inthe medium control headspace) had increased concentra-tions in HFB cells.Like in A549 cells, methacrolein, 2-methylpropanal, 3-
methylbutanal, and n-butyl acetate showed diminishedconcentrations in the headspace of HBEpC cells. Metha-crolein was not detected at all in the headspace of cellcultures, and n-butyl acetate was reduced to 19.1% of me-dium control concentration. The aldehyde 2-methylpro-panal was found only in one of three cell samples,whereas 3-methylbutanal, which was detected in allsamples measured, was reduced to 1.1% of medium con-trol concentration (Fig. 2). Moreover, acetaldehyde and
Figure 1. VOCs present at higher or lower concentrations in the headspace of A549 cells than in the medium control. Shown are average concentrations(ppbv) in logarithmic scaling with SD for 25 million cells (n = 5; dashed columns), 75 million cells (n = 4; squared columns), and 100 million cells(n = 5; crossdashed columns) compared with medium (n = 4; empty columns). *, significant differences.
Cancer Epidemiol Biomarkers Prev; 19(1) January 2010 187
NOTE: CAS numbers (Chemical Abstracts Service), correlation coefficients (R2), and LODs expressed in concentration unit [ppbv]are presented. Average concentrations (ppbv) are given with SDs. The ratio of the average concentrations of the target analytecompared with medium control and the P values of Kruskal-Wallis tests have been calculated for each cell density.
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2-butenal (below LOD in the headspace of cells) showeddecreased concentrations. Exclusively decreased inHBEpC cells were the aldehydes hexanal and octanal.In the case of the second control cell line hFB, the
concentrations of 15 compounds were increased and of
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8 were decreased (Table 2). Altogether, three independentexperiments with 50 million cells and four independentmeasurements with medium control were done. Likein A549 cancer cells, the concentrations of methyl tert-butyl ether, 2-pentanone, and 2,4-dimethyl-1-heptene
were found to be significantly increased for hFB cells(Table 2; Fig. 3).Among the remaining 12 VOCs with increased concen-
trations in hFB cells, 6 were branched saturated hydro-carbons including 2,4-dimethylhexane, which was notdetected in the medium control, 2,3,4-trimethylpentane,2,3,3-trimethylpentane, 4-methylheptane, 3-methylhep-tane, and 2,3,5-trimethylhexane (not detected in the me-dium control headspace). Increased concentrations werealso found for octane and several alcohols, such as
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2-methyl-1-propanol (below LOD in medium control),3-methyl-1-butanol (not detected in medium control),and 2-ethyl-1-hexanol (221.5% of medium control con-centration). Two unique analytes released by hFB cellswere 2-hexanone and benzene.As in A549 cells, n-butyl acetate, 2-methylpropanal,
and 3-methylbutanal were significantly decreased in theheadspace of hFB cells. Other aldehydes with decreasedconcentrations were acetaldehyde, 2-methylbutanal, 2-butenal (not detected in the headspace of cells), and
Cancer Epidemiol Biomarkers Prev; 19(1) January 2010 189
benzaldehyde. The only compound that was significantlydecreased (consumed or degraded) exclusively by hFBswas the ketone (E)-3-penten-2-one (not detected in theheadspace of cells; Table 2; Fig. 3).
Discussion
VOCs belonging to various classes of chemical com-pounds have been linked previously to lung cancer by dif-ferent authors (14, 17-21). For most of these compounds,the cellular and biochemical origin has not been deter-mined and some of them might be of exogenous origin.For the production or consumption of the compoundsfound, different types of cells could be responsible includ-ing nontumorous cells, i.e., normal surrounding tissue,immune cells, or even infectious agents. In the study pre-sented here, we attempted not only to provide further in-sight into VOCs specifically released by cancer cells(31, 32),5 but also to look for the presence of VOCs, whichmay help to discriminate normal from transformed cells.This knowledge will be essential to introduce VOCs intoroutine screening procedures. A common feature of thecell lines we have studied thus far (31, 32),5 with the
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exemption of NCI-H1666, is the fact that similar hydrocar-bons are released at significant level. In contrast, severalaldehydes and n-butyl acetate were consumed by thesecell lines. In addition, no hydrocarbon was taken up(consumed or degraded) and no aldehydes or n-butylacetate were ever released by these cells.Merely a few compounds found with the tested lung
cancer cell lines A549, NCI-H2087, and CALU-1 areunique and not found in the control cells tested here. Par-ticularly interesting among them is 2-methylpentane (re-leased by NCI-H2087), which has been detected at higherconcentrations in the breath of patients suffering fromnon–small cell lung cancer (20). 4-Methyloctane, exclu-sively found in CALU-1 cells, has been reported by Phil-lips et al. (14-16, 38) and has been used to differentiatebetween lung cancer patients and healthy volunteers.Similarly, in our work with lung cancer patients (21),we have obtained evidence that some branched hydro-carbons are important VOCs, but also alcohols and ke-tones are found to be increased in concentration inthe breath of cancer patients. Nevertheless, it should benoted that no hydrocarbon was significantly released byat least two of the different cancer cell lines studied here.This may be due to the fact that every tumor cell line
Figure 2. VOCs present at higher or lower concentrations in the headspace of the HBEpC cell line than in medium controls. Presented are averageconcentrations (ppbv) in logarithmic scaling with SD for 50 million cells (n = 3; gray columns) compared with medium (n = 4; empty columns). *, significantdifferences.
is only a limited representation of the human primarytumor it has been derived from, and only the comparisonon many more such lines and the inclusion of primarymaterial will allow to pinpoint VOCs, which can serveas biomarkers.Interestingly, more compounds with significant differ-
ences in their concentration to medium controls werereleased by healthy than tumor cells. This can be seenfor the release of the branched hydrocarbons 2,3,4-trimethylpentane, 2,4-dimethylhexane, 4-methylheptane,and 3-methylheptane, where no differences to the medi-um control were found in the cancer cell line studies (Table3). Furthermore, the differences in the concentration offour hydrocarbons, 2,3,3-trimethylpentane, n-octane,2,3,5-trimethylhexane, and 2,4-dimethyl-1-heptene, be-tween cancer cells and medium controls were higherfor nontransformed than A549 cells.Higher activity of nontransformed cells in production
of VOCs could also be suspected because of the moreabundant release of alcohols. In particular, 2-methyl-1-propanol, 2-methyl-2-propanol, and 3-methyl-1-butanolwere found to be significantly released only by healthycell lines and not by any of cancer cell lines tested in thisor earlier studies (Table 3). These data suggest that the
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metabolic pathways, which have lead to the generationof these compounds, have been more active in normalthan in transformed cells and point to a possible tumorsuppressive function. Interestingly, 2-ethyl-1-hexanol,which was released at nearly the same level by hFBand NCI-H2087 cells, was also released from cells ofthe dermis as described previously (39). The only alco-hol produced exclusively by cancer cells was ethanol(Table 1; Fig. 1). Restricted to primary bronchial cellswas the release of the esters methyl acetate and n-propylacetate. No esters at all were found to be significantlyreleased by any cancer cell line investigated in thisand previous studies (31, 32).5 However, this differencebetween carcinogenic and noncarcinogenic cells, sug-gested by our studies, needs to be confirmed by addi-tional investigations.The only two ethers significantly released by cancer
cells are methyl tert-butyl ether and ethyl tert-butyl ether(released by A549 cells). Interestingly, the highest con-centration of methyl tert-butyl ether was observed for25 million A549 cells, perhaps reflecting better growthconditions at lower cell numbers. However, becausethe concentration of methyl tert-butyl ether released isvery similar among cells, the concentration profile of
Figure 3. VOCs present at higher or lower concentrations in the headspace of the hFB cell line than in medium controls. Presented are averageconcentrations (ppbv) in logarithmic scaling with SD for 50 million cells (n = 3; gray columns) compared with medium (n = 4; empty columns). *, significantdifferences.
Cancer Epidemiol Biomarkers Prev; 19(1) January 2010 191
this analyte is most likely within the range of randomerror and the sensitivity of the applied methods is insuf-ficient to detect any increase. Both ethers were alsosignificantly released by one of the nontransformedcell types, methyl tert-butyl ether by hFB and ethyltert-butyl ether by HBEpC. In contrast, these etherswere found to be significantly decreased (degraded)by CALU-1 cancer cells.For ketones, a representative analyte was acetone. In
the experiments discussed here, acetone was releasedby nontransformed (HBEpC) and cancer (A549) cell lines.Among other ketones, 2-pentanone was secreted by allthree currently tested cell lines (hFB, HBEpC, andA549), whereas 2-hexanone was only released by hFBcells. It should be noted that as for some of previouslymentioned metabolites, hFB cells released the highestamounts of 2-pentanone. On the other hand, two otherketones were taken up (consumed or degraded), namely3-penten-2-one by hFBs and 2-butanone, by previouslyinvestigated CALU-1 cancer cells.A significant observation in this study is the strong de-
crease in the concentration of numerous aldehydes andn-butyl acetate in the headspace of A549 cell culturesand control cells (Tables 1 and 2; Figs. 1-3). Amongthe decreased VOCs, the ester n-butyl acetate and thealdehyde 3-methylbutanal were found to be lowered inall tested cell lines. Previous work on NCI-H2087 (32),NCI-H1666,5 and CALU-1 (31) lung cancer cell linesshowed that both 3-methylbutanal and n-butyl acetatewere decreased in all cells investigated in vitro. Typically,but not always, the aldehydes acetaldehyde, 2-methyl-propanal, and methacrolein were also found to be de-creased (Table 3). This decrease could be observedeither in the lung cancer cell lines A549, NCI-H2087,or CALU-1, or in one of the tested control cell lines(Table 3; Figs. 1-3). 2-Ethylacrolein and 2-ethyl-2-butenalwere only degraded by lung cancer cell lines. A HBEpC-specific feature not found in any of the other investigatedcell lines was the degradation of n-octanal, whereas
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only hFB cells showed a decrease in 3-penten-2-one. Itshould be noticed that besides the ester n-butyl acetate(and 3-penten-2-one for hFB), nontransformed cells onlydegrade aldehydes, whereas cancer cells could alsodegrade nitrogen-containing compounds (pyrrole byA549 and acetonitrile by CALU-1), ketone, and ethers(acetone, methyl tert-butyl ether, and ethyl tert-butylether, respectively, all degraded by the CALU-1 line).Overall, the reasons for differences in VOC release
or consumption among the investigated cell lines arecurrently unknown, but may result from phenotypicor genotypic differences. Clarification of this issue willrequire an understanding of the underlying molecularmechanisms for VOC production, which is currentlylacking for the mentioned compounds.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank the Member of the Tyrolean regional govern-ment Dr. Erwin Koler and the Director of the UniversityClinic of Innsbruck (TILAK) Mag. Andreas Steiner fortheir generous support.
Grant Support
European Commission (project BAMOD, project NoLSHC-CT-2005-019031) and the Austrian Ministry forScience and Research (BMWF, Vienna, Austria, grantsGZ 651.019/1-VI/2/2006, GZ 651.019/3-VI/2/2006).The costs of publication of this article were defrayed
in part by the payment of page charges. This article musttherefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.Received 2/20/09; revised 10/13/09; accepted 11/3/09;
published online 1/7/10.
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