Factors that influence the quality of metabolomics data in in vitro cell toxicity studies: a systematic survey

AbstractREACH (Registration, Evaluation, Authorization and Restriction of Chemicals) is a global strategy and regulation policy of the EU that aims to improve the protection of human health and the environment through the better and earlier identification of the intrinsic properties of chemical substances. It entered into force on 1st June 2007 (EC 1907/2006). REACH and EU policies plead for the use of robust high-throughput "omic" techniques for the in vitro investigation of the toxicity of chemicals that can provide an estimation of their hazards as well as information regarding the underlying mechanisms of toxicity. In agreement with the 3R’s principles, cultured cells are nowadays widely used for this purpose, where metabolomics can provide a real-time picture of the metabolic effects caused by exposure of cells to xenobiotics, enabling the estimations about their toxicological hazards. High quality and robust metabolomics data sets are essential for precise and accurate hazard predictions. Currently, the acquisition of consistent and representative metabolomic data is hampered by experimental drawbacks that hinder reproducibility and difficult robust hazard interpretation. Using the differentiated human liver HepG2 cells as model system, and incubating with hepatotoxic (acetaminophen and valproic acid) and non-hepatotoxic compounds (citric acid), we evaluated in-depth the impact of several key experimental factors (namely, cell passage, processing day and storage time, and compound treatment) and instrumental factors (batch effect) on the outcome of an UPLC-MS metabolomic analysis data set. Results showed that processing day and storage time had a significant impact on the retrieved cell's metabolome, while the effect of cell passage was minor. Meta-analysis of results from pathway analysis showed that batch effect corrections and quality control (QC) measures are critical to enable consistent and meaningful estimations of the effects caused by compounds on cells. The quantitative analysis of the changes in metabolic pathways upon bioactive compound treatment remained consistent despite the concurrent causes of metabolomic data variation. Thus, upon appropriate data retrieval and correction and by an innovative metabolic pathway analysis, the metabolic alteration predictions remained conclusive despite the acknowledged sources of variability.

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Factors that Influence the Quality of Metabolomics Data in in Vitro Cell Toxicity Studies: A Systematic Survey

10.21203/rs.3.rs-724307/v1 ◽

2021 ◽

Author(s):

Marta Moreno-Torres ◽

Guillem García-Llorens ◽

Erika Moro ◽

Rebeca Méndez ◽

Guillermo Quintás ◽

...

Keyword(s):

Pathway Analysis ◽

Storage Time ◽

Data Retrieval ◽

Batch Effect ◽

Metabolic Effects ◽

Cell Passage ◽

Metabolomics Data ◽

Metabolomic Data ◽

And Storage

Abstract REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) is a global strategy and regulation policy of the EU that aims to improve the protection of human health and the environment through the better and earlier identification of the intrinsic properties of chemical substances. It entered into force on 1st June 2007 (EC 1907/2006). REACH and EU policies plead for the use of robust high-throughput "omic" techniques for the in vitro investigation of the toxicity of chemicals that can provide an estimation of their hazards as well as information regarding the underlying mechanisms of toxicity. In agreement with the 3R’s principles, cultured cells are nowadays widely used for this purpose, where metabolomics can provide a real-time picture of the metabolic effects caused by exposure of cells to xenobiotics, enabling the estimations about their toxicological hazards. High quality and robust metabolomics data sets are essential for precise and accurate hazard predictions. Currently, the acquisition of consistent and representative metabolomic data is hampered by experimental drawbacks that hinder reproducibility and difficult robust hazard interpretation. Using the differentiated human liver HepG2 cells as model system, and incubating with hepatotoxic (acetaminophen and valproic acid) and non-hepatotoxic compounds (citric acid), we evaluated in-depth the impact of several key experimental factors (namely, cell passage, processing day and storage time, and compound treatment) and instrumental factors (batch effect) on the outcome of an UPLC-MS metabolomic analysis data set. Results showed that processing day and storage time had a significant impact on the retrieved cell's metabolome, while the effect of cell passage was minor. Meta-analysis of results from pathway analysis showed that batch effect corrections and quality control (QC) measures are critical to enable consistent and meaningful estimations of the effects caused by compounds on cells. The quantitative analysis of the changes in metabolic pathways upon bioactive compound treatment remained consistent despite the concurrent causes of metabolomic data variation. Thus, upon appropriate data retrieval and correction and by an innovative metabolic pathway analysis, the metabolic alteration predictions remained conclusive despite the acknowledged sources of variability.

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The influence of preparation and storage time on endothelial cells in Quarter–Descemet membrane endothelial keratoplasty (Quarter–DMEK) grafts in vitro

Cell and Tissue Banking ◽

10.1007/s10561-020-09854-z ◽

2020 ◽

Vol 21 (4) ◽

pp. 615-623

Author(s):

Daniele Spinozzi ◽

Alina Miron ◽

Jessica Lie ◽

Silke Oellerich ◽

Sorcha Ni Dhubhghaill ◽

...

Keyword(s):

Endothelial Cells ◽

Storage Time ◽

Endothelial Keratoplasty ◽

Descemet Membrane Endothelial Keratoplasty ◽

Descemet Membrane ◽

And Storage

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Reliability of the Heparin Management Test for Monitoring High Levels of Unfractionated Heparin

Anesthesiology ◽

10.1097/00000542-200006000-00016 ◽

2000 ◽

Vol 92 (6) ◽

pp. 1594-1602 ◽

Cited By ~ 8

Author(s):

Fritz Mertzlufft ◽

Andreas Koster ◽

Roland Hansen ◽

Anne Risch ◽

Herrmann Kuppe ◽

...

Keyword(s):

Cardiopulmonary Bypass ◽

Storage Time ◽

Coagulation Factors ◽

Clotting Time ◽

Activated Clotting Time ◽

Group I ◽

Group Ii ◽

And Storage

Background The authors assessed the heparin management test in vitro in volunteers and in vivo during cardiopulmonary bypass. Methods In vitro, the heparin management test was analyzed for heparin levels between 0 and 6 IU/ml using variations in hematocrit, platelets, procoagulants, and storage time. The in vivostudies consisted of two groups: In group I (cardiopulmonary bypass </= 90 min, n = 40), anticoagulation was performed according to the activated clotting time (with or without aprotinin); in group II (cardiopulmonary bypass >/= 180 min, with aprotinin) included use (n = 10) and nonuse of coumadin (n = 10) and anticoagulation according to the automated heparin dose-response assay. Tests were performed in duplicate (whole blood, two heparin management test analyzers) and compared with anti-Xa activity (plasma). Results In vitro, the results of the heparin management test (n = 1,070) correlated well with heparin concentration (r2 = 0.98). Dilution and storage time did not affect the heparin management test; a hematocrit of 60% and reduced procoagulants (10%) prolonged clotting time. In vivo, the correlation (heparin management test vs. anti-Xa) was strong in group I (r2 = 0.97 [with aprotinin] and 0.96 [without aprotinin]; n = 960) and group II without coumadin (r2 = 0.89, n = 516). In group II with coumadin, the overall correlation was r2 = 0.87 and 0.79 (n = 484), although the range varied widely (0.57-0.94, between-analyzer differences 0-47%). Conclusions The results of the heparin management test were influenced by hematocrit, plasma coagulation factors, and the heparin level, but not by use of aprotinin. The heparin management test provided reliable values in vitro in group I, and in group II without coumadin but was less reliable in group II with coumadin.

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Pollen viability in Kunzea pomifera (Myrtaceae) as influenced by sucrose concentration and storage

Australian Journal of Botany ◽

10.1071/bt04054 ◽

2006 ◽

Vol 54 (6) ◽

pp. 553 ◽

Cited By ~ 2

Author(s):

T. Page ◽

G. M. Moore ◽

J. Will ◽

G. M. Halloran

Keyword(s):

Pollen Germination ◽

Storage Time ◽

Sucrose Concentration ◽

Pollen Viability ◽

Germination Percentage ◽

Liquid Media ◽

Controlled Pollination ◽

Fresh Pollen ◽

And Storage

In vitro germination of freshly collected pollen was examined for five genotypes of Kunzea pomifera in liquid media with 5% and 10% sucrose. There were no significant differences in germination level between the different sucrose concentrations for each of the genotypes. Pollen-germination levels were significantly different among genotypes, ranging from 7 to 89%. Pollen of one genotype, stored for 12 months at 4°C and 10% relative humidity, was evaluated for germination at intervals over this period, and while significant differences in the levels of pollen germination after different storage periods were found, there was no correlation between germination percentage and storage time. Pollen of the K. pomifera accession Kmt1 stored under such conditions for 370 days was used in controlled pollination with accession Kmt2, resulting in 3.4 seeds per fruit, which was significantly fewer than the 17.2 seeds per fruit when using fresh pollen.

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