Summary of Study ST002543
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001638. The data can be accessed directly via it's Project DOI: 10.21228/M8ZX4D This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST002543 |
| Study Title | GC/MS analysis of hypoxic volatile metabolic markers in the MDA-MB-231 breast cancer cell line |
| Study Summary | Hypoxia in disease describes persistent low oxygen conditions, observed in a range of pathologies, including cancer. In the discovery of biomarkers in biological models, pathophysiological traits present a source of translatable metabolic products for the diagnosis of disease in humans. Part of the metabolome is represented by its volatile, gaseous fraction; the volatilome. Human volatile profiles, such as those found in breath, are able to diagnose disease, however accurate volatile biomarker discovery is required to target reliable biomarkers to develop new diagnostic tools. Using custom chambers to control oxygen levels and facilitate headspace sampling, the MDA-MB-231 breast cancer cell line was exposed to hypoxia (1% oxygen) for 24 hours. The maintenance of hypoxic conditions in the system was successfully validated over this time period. Targeted and untargeted gas chromatography mass spectrometry approaches revealed four significantly altered volatile organic compounds when compared to control cells. Three compounds were actively consumed by cells: methyl chloride, acetone and n-Hexane. Cells under hypoxia also produced significant amounts of styrene. This work presents a novel methodology for identification of volatile metabolisms under controlled gas conditions with novel observations of volatile metabolisms by breast cancer cells. |
| Institute | University of York |
| Last Name | Issitt |
| First Name | Theo |
| Address | Biology Dept. University of York, Personal |
| ti538@york.ac.uk | |
| Phone | 07398244497 |
| Submit Date | 2023-03-31 |
| Num Groups | 4 |
| Publications | T. Issitt et al., Volatile compounds in human breath: critical review and meta-analysis Journal of Breath Research, Volume 16, Number 2 (2022) https://iopscience.iop.org/article/10.1088/1752-7163/ac5230#jbrac5230s2 |
| Analysis Type Detail | GC-MS |
| Release Date | 2023-04-21 |
| Release Version | 1 |
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Treatment:
| Treatment ID: | TR002655 |
| Treatment Summary: | Cells were placed in static headspace chambers as previously described [4] with new, clean silicon gaskets. Low oxygen, hypoxic gas (1 % O2, 5 % CO2, 94 % N2; purchased from BOC Specialty Gases, Woking, UK) was flushed through the chambers at a rate of 4 L/min for 10 min (chamber volume = 25 L). Chambers were then closed and placed at 37 ˚C for 2 hours to allow residual oxygen in the media to equilibrate with chamber headspace. Chambers were then flushed again at a rate of 4 L/min for 10 min, sealed and returned to 37 ˚C. After a further 24 hours, chambers were flushed again at a rate of 4 L/min for 10 min. 15 ml of gas standards (MeCl, 520 ppb (parts per billion); MeBr, 22 ppb; MeI, 26 ppb; DMS, 110 ppb; CFC-11, 400 ppb and CH3Cl3, 110ppb; BOC Specialty Gases, Woking, UK) were then injected into the chambers through a butyl seal and time zero sample taken. Injected compounds are either known metabolites for cancer cells, or internal standards (CFC-11) for the analysis and quantification of metabolism. Final chamber concentrations were similar to environmental concentrations, e.g MeCl, 1.2 ppb and MeBr 0.05 ppb, particularly more polluted urban spaces (Redeker et al., 2007). Injected gases are the same as those used for calibration. Compounds not injected but detected at first time point, due to residual presence from laboratory air, (including isoprene, acetone, 2-MP, 3-MP and n-hexane) were quantified. Two time zero (T0) samples were taken using an evacuated 500 mL electropolished stainless steel canister (LabCommerce, San Jose, USA) through fine mesh Ascarite® traps (Archbold et al., 2005), after which the chamber was resealed and left on a platform rocker on its slowest setting for 120 min, at which point two further air samples (T1) were collected. Duplicate samples were taken so that two analytical approaches could be performed (targeted and non-untargeted MS). Cells were removed from the chamber, washed with PBS twice and lysed in 500 µL RIPA buffer (NaCl (5 M), 5 mL Tris-HCl (1 M, pH 8.0), 1 mL Nonidet P-40, 5 mL sodium deoxycholate (10 %), 1 mL SDS (10 %)) with protease inhibitor (Sigma-Aldrich, Roche; Mannheim, Germany). Protein concentration of lysates were determined using BCA assay (Thermo Scientific, Waltham, MA, USA). Media alone was treated exactly the same as cells, and only acetone was found to differ significantly between conditions (Supplementary figure 1). These media blank outcomes were subtracted from respective cellular samples prior to protein normalisation. Comparative controls include lab air blanks and those data available from the dataset and collection method published previously which created and quantified metabolic fluxes of volatile compounds from MDA-MB-231 under hyperoxic (lab air) conditions (Issitt et al., 2022a). |
| Treatment: | Hypoxia |
| Treatment Vehicle: | Nitrogen |
| Cell Storage: | 37 degrees |
| Cell Media: | DMEM |
| Cell Envir Cond: | Hypoxia/lab air |
