Summary of Study ST001007

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 PR000681. The data can be accessed directly via it's Project DOI: 10.21228/M8NH4G This work is supported by NIH grant, U2C- DK119886.

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

Study ID	ST001007
Study Title	Multi-Platform Physiologic and Metabolic Phenotyping Reveals Microbial Toxicity (part-I)
Study Summary	The gut microbiota are susceptible to modulation by environmental stimuli and therefore can serve as biological sensors. Recent evidence suggests that xenobiotics can disrupt the interaction between the microbiota and host. Here, we describe an approach that combines in vitro microbial incubation (isolated cecal contents from mice), flow cytometry, and mass spectrometry- and 1H NMR-based metabolomics to evaluate xenobiotic-induced microbial toxicity. Tempol, a stabilized free radical scavenger known to remodel the microbial community structure and function in vivo, was studied to assess its direct effects on the gut microbiota. Microbiota were isolated from mouse cecum and were exposed to tempol for 4 h under strict anaerobic condition. The flow cytometry data suggested short term exposure of the microbiota to tempol is associated with disrupted membrane physiology as well as compromised metabolic activity. Mass spectrometry and NMR metabolomics revealed that tempol exposure significantly disrupted microbial metabolic activity, specifically indicated by changes in short chain fatty acids, branched chain amino acids, amino acids, nucleotides, glucose, and oligosaccharides. In addition, a mouse study with tempol (5 days gavage) showed similar microbial physiologic and metabolic changes, indicating the in vitro approach reflected in vivo conditions. Our results, through evaluation of microbial viability, physiology and metabolism, and comparison of in vitro and in vivo exposures with tempol, suggests that physiologic and metabolic phenotyping provides unique insight into gut microbiota toxicity.
Institute	Pennsylvania State University
Last Name	Nichols
First Name	Robert
Address	650 Toftrees ave
Email	rgn5011@psu.edu
Phone	7247662694
Submit Date	2018-07-13
Raw Data File Type(s)	fid
Analysis Type Detail	NMR
Release Date	2018-08-27
Release Version	1

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Project:

Project ID:	PR000681
Project DOI:	doi: 10.21228/M8NH4G
Project Title:	Multi-Platform Physiologic and Metabolic Phenotyping Reveals Microbial Toxicity
Project Summary:	The gut microbiota are susceptible to modulation by environmental stimuli and therefore can serve as biological sensors. Recent evidence suggests that xenobiotics can disrupt the interaction between the microbiota and host. Here, we describe an approach that combines in vitro microbial incubation (isolated cecal contents from mice), flow cytometry, and mass spectrometry- and 1H NMR-based metabolomics to evaluate xenobiotic-induced microbial toxicity. Tempol, a stabilized free radical scavenger known to remodel the microbial community structure and function in vivo, was studied to assess its direct effects on the gut microbiota. Microbiota were isolated from mouse cecum and were exposed to tempol for 4 h under strict anaerobic condition. The flow cytometry data suggested short term exposure of the microbiota to tempol is associated with disrupted membrane physiology as well as compromised metabolic activity. Mass spectrometry and NMR metabolomics revealed that tempol exposure significantly disrupted microbial metabolic activity, specifically indicated by changes in short chain fatty acids, branched chain amino acids, amino acids, nucleotides, glucose, and oligosaccharides. In addition, a mouse study with tempol (5 days gavage) showed similar microbial physiologic and metabolic changes, indicating the in vitro approach reflected in vivo conditions. Our results, through evaluation of microbial viability, physiology and metabolism, and comparison of in vitro and in vivo exposures with tempol, suggests that physiologic and metabolic phenotyping provides unique insight into gut microbiota toxicity.
Institute:	Pennsylvania State University
Last Name:	Nichols
First Name:	Robert
Address:	650 toftrees ave Apt #108, State College, Pa 16802
Email:	rgn5011@psu.edu
Phone:	7247662694

Subject:

Subject ID:	SU001046
Subject Type:	Other
Subject Species:	Mus musculus
Taxonomy ID:	10090
Species Group:	Microbiome

Factors:

Subject type: Other; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment
SA063408	C1	Control
SA063409	C6	Control
SA063410	C5	Control
SA063411	C2	Control
SA063412	C3	Control
SA063413	C4	Control
SA063414	H6	High-Dose Tempol
SA063415	H5	High-Dose Tempol
SA063416	H1	High-Dose Tempol
SA063417	H3	High-Dose Tempol
SA063418	H4	High-Dose Tempol
SA063419	H2	High-Dose Tempol
SA063420	L4	Low-Dose Tempol
SA063421	L1	Low-Dose Tempol
SA063422	L2	Low-Dose Tempol
SA063423	L3	Low-Dose Tempol
SA063424	L5	Low-Dose Tempol
SA063425	L6	Low-Dose Tempol
SA063426	M1	Medium-Dose-Tempol
SA063427	M2	Medium-Dose-Tempol
SA063428	M3	Medium-Dose-Tempol
SA063429	M4	Medium-Dose-Tempol
SA063430	M5	Medium-Dose-Tempol
SA063431	M6	Medium-Dose-Tempol

Showing results 1 to 24 of 24

Showing results 1 to 24 of 24

Collection:

Collection ID:	CO001040
Collection Summary:	6-week-old wild-type male C57BL/6J mice (Jackson Laboratory, Bar Harbor, Maine) were transferred into anaerobic chamber (Coy Laboratory Products, Inc., Grass Lake, MI) following CO2 asphyxiation. All the following procedures were performed under strict anaerobic conditions with an oxygen level below 20 ppm and cecal contents were collected
Sample Type:	Cecum

Treatment:

Treatment ID:	TR001060
Treatment Summary:	The cecal content suspension was treated with tempol at a final concentration 0.01 mg/mL, 0.1 mg/mL and 1 mg/mL, following a brief centrifugation and incubation at 37 °C for 4 h in dark.

Sample Preparation:

Sampleprep ID:	SP001053
Sampleprep Summary:	The microbiota suspension saved after 4h incubation was used for 1H NMR spectroscopy. 1 mL of microbiota suspension was centrifuged at low speed (700 g, 4 °C for 1 min) to pellet down large particles. The maximum supernatant volume was transferred to a new tube, centrifuged at high speed (6000 g, 4 °C for 3 min) to pellet down bacteria. The microbial pellet was washed two times with PBS. After the third wash, 1 mL of pre-cooled methanol:H2O (v/v = 2:1) and 1.0 mm diameter zirconia/silica beads (BioSpec, Bartlesville, OK) were added to the microbial pellet, followed by homogenization (6500 rpm, 1 cycle, 60 s) using the Precellys tissue homogenizer (Bertin Technologies, Rockville, MD). The homogenized sample was freeze-thawed three times with liquid nitrogen and 37°C water bath, then was homogenized again and sonicated for 15 min at 250W with Branson 1510 Ultrasonic Cleaner (Branson Ultrasonics, Danbury, CT) to rupture microbial cell walls and release intracellular metabolites. The sample was centrifuged (11180g, 4 °C, and 10 min) and the supernatants was transferred to a new 2 mL tube. Another 1 mL methanol:H2O (v/v = 2:1) was added to the pellets and the extraction procedure was repeated. All supernatants were combined, dried down and reconstituted in 600 μL of PBS (K2HPO4/NaH2PO4, 0.1M, pH 7.4, containing 50% D2O and 0.005% TSP-d4 as internal standard). Following centrifugation (13 000g, 4 °C, 10min), 550 μL of each extract was transferred into a 5 mm NMR tube for analysis.

Sampleprep ID:

SP001053

Sampleprep Summary:

The microbiota suspension saved after 4h incubation was used for 1H NMR spectroscopy. 1 mL of microbiota suspension was centrifuged at low speed (700 g, 4 °C for 1 min) to pellet down large particles. The maximum supernatant volume was transferred to a new tube, centrifuged at high speed (6000 g, 4 °C for 3 min) to pellet down bacteria. The microbial pellet was washed two times with PBS. After the third wash, 1 mL of pre-cooled methanol:H2O (v/v = 2:1) and 1.0 mm diameter zirconia/silica beads (BioSpec, Bartlesville, OK) were added to the microbial pellet, followed by homogenization (6500 rpm, 1 cycle, 60 s) using the Precellys tissue homogenizer (Bertin Technologies, Rockville, MD). The homogenized sample was freeze-thawed three times with liquid nitrogen and 37°C water bath, then was homogenized again and sonicated for 15 min at 250W with Branson 1510 Ultrasonic Cleaner (Branson Ultrasonics, Danbury, CT) to rupture microbial cell walls and release intracellular metabolites. The sample was centrifuged (11180g, 4 °C, and 10 min) and the supernatants was transferred to a new 2 mL tube. Another 1 mL methanol:H2O (v/v = 2:1) was added to the pellets and the extraction procedure was repeated. All supernatants were combined, dried down and reconstituted in 600 μL of PBS (K2HPO4/NaH2PO4, 0.1M, pH 7.4, containing 50% D2O and 0.005% TSP-d4 as internal standard). Following centrifugation (13 000g, 4 °C, 10min), 550 μL of each extract was transferred into a 5 mm NMR tube for analysis.

Analysis:

Analysis ID:	AN001649
Analysis Type:	NMR
Num Factors:	4
Num Metabolites:	14
Units:	ppm

NMR:

NMR ID:	NM000124
Analysis ID:	AN001649
Instrument Name:	Bruker
Instrument Type:	FT-NMR
NMR Experiment Type:	1D 1H
Spectrometer Frequency:	600 MHz