Summary of Study ST002157

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 PR001370. The data can be accessed directly via it's Project DOI: 10.21228/M8N12W This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002157
Study TitleEffect of long-term exposure to graphene on skin cell metabolism
Study SummaryGraphene-derived materials are a family of nanomaterials with multiple potential applications in different fields such as biomedicine. It is therefore essential to understand their interaction with cellular barriers such as skin. In this work we evaluated the metabolic changes in human skin cells (HaCaT) exposed to different GRMs for 7 and 30 days. Objectives Endogenous metabolic profiles of control and graphene-treated keratinocytes have been studied using ultra-high performance liquid chromatography – mass spectrometry (UHPLC-MS). Keratinocytes were treated with graphene oxide (GO) from two different suppliers and with few layer graphene (FLG). Samples were collected one week and one month after the start of the treatment. The general aim of the project was to evaluate potential metabolic differences between: 1) Graphene-treated keratinocytes and control keratinocytes at one week; 2) Graphene-treated keratinocytes and control keratinocytes at one month; 3) Control keratinocytes at 1 month and 1 week; 4) Graphene-treated keratinocytes at 1 month and 1 week. Experimental Procedures A successful metabolic profiling experiment relies on the ability to determine changes in an organism’s biofluid or tissue complement of metabolites. Mass spectrometry coupled to ultra-high performance liquid chromatography (UHPLC-MS) is well suited to such analyses due to its high sensitivity, large coverage over different classes of metabolites, high throughput capacity, and wide dynamic range. In this study, one UHPLC-MS based platform was used to analyse endogenous analytes for inclusion in subsequent statistical analysis procedures used to study metabolic differences between the groups of samples. Results The oxidation degree and size of the GRMs is determinant in the effect on cell metabolism, as well as the exposure time. Thus, one of the materials used generated a change in the energy metabolism of the cells, significantly increasing the level of different Krebs cycle metabolites.
Institute
University of Castilla-La Mancha
DepartmentMedical Sciences
LaboratoryGroup of Oxidative Stress and Neurodegeneration
Last NameFrontinan
First NameJavier
AddressF. Medicina Camino de Moledores s/n 13071
Emailjavier.frontinan@uclm.es
Phone+34656967979
Submit Date2022-04-25
Num Groups8
Raw Data AvailableYes
Raw Data File Type(s)raw(Waters)
Analysis Type DetailLC-MS
Release Date2022-06-01
Release Version1
Javier Frontinan Javier Frontinan
https://dx.doi.org/10.21228/M8N12W
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001370
Project DOI:doi: 10.21228/M8N12W
Project Title:Effect of long-term exposure to graphene on skin cell metabolism
Project Type:MS HaCaT Cells
Project Summary:Graphene-derived materials are a family of nanomaterials with multiple potential applications in different fields such as biomedicine. It is therefore essential to understand their interaction with cellular barriers such as skin. In this work we evaluated the metabolic changes in human skin cells (HaCaT) exposed to different GRMs for 7 and 30 days. Objectives Endogenous metabolic profiles of control and graphene-treated keratinocytes have been studied using ultra-high performance liquid chromatography – mass spectrometry (UHPLC-MS). Keratinocytes were treated with graphene oxide (GO) from two different suppliers and with few layer graphene (FLG). Samples were collected one week and one month after the start of the treatment. The general aim of the project was to evaluate potential metabolic differences between: 1) Graphene-treated keratinocytes and control keratinocytes at one week; 2) Graphene-treated keratinocytes and control keratinocytes at one month; 3) Control keratinocytes at 1 month and 1 week; 4) Graphene-treated keratinocytes at 1 month and 1 week. Experimental Procedures A successful metabolic profiling experiment relies on the ability to determine changes in an organism’s biofluid or tissue complement of metabolites. Mass spectrometry coupled to ultra-high performance liquid chromatography (UHPLC-MS) is well suited to such analyses due to its high sensitivity, large coverage over different classes of metabolites, high throughput capacity, and wide dynamic range. In this study, one UHPLC-MS based platform was used to analyse endogenous analytes for inclusion in subsequent statistical analysis procedures used to study metabolic differences between the groups of samples.
Institute:University of Castilla-La Mancha
Department:Medical Sciences
Laboratory:Group of Oxidative Stress and Neurodegeneration
Last Name:Frontiñán
First Name:Javier
Address:F. Medicina Camino de Moledores s/n 13071
Email:javier.frontinan@uclm.es
Phone:+34656967979
Funding Source:Graphene Flagship (EU)

Subject:

Subject ID:SU002243
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Gender:Male
Cell Biosource Or Supplier:CLS
Cell Strain Details:In vitro spontaneously transformed keratinocytes from histologically normal skin
Cell Primary Immortalized:HaCaT
Cell Passage Number:Up to the 15th passage

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Exposure time Nanomaterial Replicate
SA206929190905_19_0009_UCLM_CCM_059Month C N1
SA206928190905_19_0009_UCLM_CCM_043Month C- N1
SA206930190905_19_0009_UCLM_CCM_049Month C N2
SA206931190905_19_0009_UCLM_CCM_050Month C N3
SA206932190905_19_0009_UCLM_CCM_030Month C N4
SA206933190905_19_0009_UCLM_CCM_060Month C N5
SA206935190905_19_0009_UCLM_CCM_024Month FLG N1
SA206934190905_19_0009_UCLM_CCM_062Month FLG- N1
SA206936190905_19_0009_UCLM_CCM_053Month FLG N2
SA206937190905_19_0009_UCLM_CCM_052Month FLG N3
SA206938190905_19_0009_UCLM_CCM_013Month FLG N4
SA206939190905_19_0009_UCLM_CCM_038Month FLG N5
SA206941190905_19_0009_UCLM_CCM_042Month GO g N1
SA206940190905_19_0009_UCLM_CCM_031Month GO g- N1
SA206942190905_19_0009_UCLM_CCM_028Month GO g N2
SA206943190905_19_0009_UCLM_CCM_064Month GO g N3
SA206944190905_19_0009_UCLM_CCM_022Month GO g N4
SA206945190905_19_0009_UCLM_CCM_035Month GO g N5
SA206947190905_19_0009_UCLM_CCM_041Month GO N1
SA206946190905_19_0009_UCLM_CCM_018Month GO- N1
SA206948190905_19_0009_UCLM_CCM_029Month GO N2
SA206949190905_19_0009_UCLM_CCM_069Month GO N3
SA206950190905_19_0009_UCLM_CCM_055Month GO N4
SA206951190905_19_0009_UCLM_CCM_019Month GO N5
SA206905190905_19_0009_UCLM_CCM_001- - -
SA206906190905_19_0009_UCLM_CCM_037- - -
SA206907190905_19_0009_UCLM_CCM_027- - -
SA206908190905_19_0009_UCLM_CCM_026- - -
SA206909190905_19_0009_UCLM_CCM_017- - -
SA206910190905_19_0009_UCLM_CCM_046- - -
SA206911190905_19_0009_UCLM_CCM_047- - -
SA206912190905_19_0009_UCLM_CCM_067- - -
SA206913190905_19_0009_UCLM_CCM_066- - -
SA206914190905_19_0009_UCLM_CCM_057- - -
SA206915190905_19_0009_UCLM_CCM_056- - -
SA206916190905_19_0009_UCLM_CCM_016- - -
SA206917190905_19_0009_UCLM_CCM_036- - -
SA206918190905_19_0009_UCLM_CCM_007- - -
SA206919190905_19_0009_UCLM_CCM_008- - -
SA206920190905_19_0009_UCLM_CCM_009- - -
SA206921190905_19_0009_UCLM_CCM_010- - -
SA206922190905_19_0009_UCLM_CCM_006- - -
SA206923190905_19_0009_UCLM_CCM_005- - -
SA206924190905_19_0009_UCLM_CCM_002- - -
SA206925190905_19_0009_UCLM_CCM_003- - -
SA206926190905_19_0009_UCLM_CCM_004- - -
SA206927190905_19_0009_UCLM_CCM_011- - -
SA206953190905_19_0009_UCLM_CCM_020Week C N1
SA206952190905_19_0009_UCLM_CCM_048Week C- N1
SA206954190905_19_0009_UCLM_CCM_070Week C N2
SA206955190905_19_0009_UCLM_CCM_061Week C N3
SA206956190905_19_0009_UCLM_CCM_025Week C N4
SA206957190905_19_0009_UCLM_CCM_039Week C N5
SA206959190905_19_0009_UCLM_CCM_014Week FLG N1
SA206958190905_19_0009_UCLM_CCM_032Week FLG- N1
SA206960190905_19_0009_UCLM_CCM_054Week FLG N2
SA206961190905_19_0009_UCLM_CCM_044Week FLG N3
SA206962190905_19_0009_UCLM_CCM_058Week FLG N4
SA206963190905_19_0009_UCLM_CCM_051Week FLG N5
SA206965190905_19_0009_UCLM_CCM_023Week GO g N1
SA206964190905_19_0009_UCLM_CCM_021Week GO g- N1
SA206966190905_19_0009_UCLM_CCM_065Week GO g N2
SA206967190905_19_0009_UCLM_CCM_068Week GO g N3
SA206968190905_19_0009_UCLM_CCM_034Week GO g N4
SA206969190905_19_0009_UCLM_CCM_033Week GO g N5
SA206971190905_19_0009_UCLM_CCM_012Week GO N1
SA206970190905_19_0009_UCLM_CCM_045Week GO- N1
SA206972190905_19_0009_UCLM_CCM_071Week GO N2
SA206973190905_19_0009_UCLM_CCM_015Week GO N3
SA206974190905_19_0009_UCLM_CCM_040Week GO N4
SA206975190905_19_0009_UCLM_CCM_063Week GO N5
Showing results 1 to 71 of 71

Collection:

Collection ID:CO002236
Collection Summary:HaCaT cells treated with GO1, GO2 and FLG
Sample Type:Keratinocytes
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002255
Treatment Summary:Cells were treated with 5 ug/mL of the different GRMs for 7 days and 30 days (1 treatment/week)

Sample Preparation:

Sampleprep ID:SP002249
Sampleprep Summary:Keratinocytes were defrosted on ice and proteins were precipitated from the lysed cell samples by adding the extraction solvent spiked with metabolites not detected in unspiked cell extracts. These metabolites, considered as internal standards, were tryptophan-d5, Anthranilic acid-(ring-13C6), Phenylthiohydantoin (PTH)-valine, Glycocholic-2,2,4,4-d4 acid (Sigma Aldrich). Then, cell extracts were incubated at -20 ˚C for 1 hour and after that, samples were vortexed and centrifuged at 18,000 x g for 10 minutes at 4 ºC. Supernatants were collected and kept on ice. A second extraction was performed from the remaining pellets, following the same steps described above. Supernatants obtained from the second extraction were collected and put together with the supernatants of the first extraction. Finally, these supernatants were dried under vacuum, reconstituted in water, resuspended with agitation for 10 minutes, centrifuged at 18,000 x g for 5 minutes at 4ºC, and transferred to vials for UHPLC-MS analysis.

Combined analysis:

Analysis ID AN003532
Analysis type MS
Chromatography type Ion pair
Chromatography system Waters Acquity H-Class
Column Waters Acquity BEH HSS T3 (100 x 2.1mm, 1.8um)
MS Type ESI
MS instrument type Time of flight
MS instrument name Waters LCT premier
Ion Mode NEGATIVE
Units Log2 (fold-change)

Chromatography:

Chromatography ID:CH002609
Chromatography Summary:Chromatography was performed on a 1.0-mm internal diameter x 150 mm Acquity HSS T3 1.7 µm column (Waters Corp., Milford, MA) using an ACQUITY UPLC system (Waters Corp.). The column was maintained at 40 ºC. Samples (2µL) were injected onto the column at a flow rate of 100 µL/min, for a total run time of 25 min. The following linear elution gradient was used: 100% solvent A (10mM tributylamine + 15mM acetic acid + 0.2% methanol in water), to which solvent B (methanol) was added incrementally to reach a concentration of 4% B after 1.5 min, increasing to 20% B at 3 min, 25% B at 8 min, 50% B at 10 min, 55% at 15 min, and 100% B over the next 5 min, and returning to the initial composition over the final 5 min.
Instrument Name:Waters Acquity H-Class
Column Name:Waters Acquity BEH HSS T3 (100 x 2.1mm, 1.8um)
Chromatography Type:Ion pair

MS:

MS ID:MS003290
Analysis ID:AN003532
Instrument Name:Waters LCT premier
Instrument Type:Time of flight
MS Type:ESI
MS Comments:The eluent was introduced into the mass spectrometer (LCT Premier (Waters Corp.) by electrospray ionization, with capillary and cone voltages set in the negative ion mode to 2800 and 100 V, respectively. The nebulization gas was set to 600 L/hour and 300ºC. The cone gas was fixed at 50 L/hour, and the source temperature was maintained at 120ºC. Centroid data were acquired over the mass range of 50-1000 Da, using an accumulation time of 0.2 seconds per spectrum. All spectra were mass corrected in real time by reference to leucine enkephalin, infused at 10 µL/minute through an independent reference electrospray, sampled every 10 seconds.
Ion Mode:NEGATIVE
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