Nontargeted Metabolite Profiling of Human Lung Epithelial Cells (A549) with HILIC Mode UPLC HRMS: Silica Nanoparticle Mediated Cytotoxicity Effects
Kalyan Paila, Pemiscot Memorial Hospital, Hayti, Missouri, MO Shyam Arvamudhan, NC A and T University, Greensboro, NC
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Kalyan Paila (Presenter) Pemiscot Memorial Hospital
Presenter Bio: I'm happy to introduce myself as Kalyan Paila,working in a Scientist role in Pemiscot Memorial Hospital in southern Missouri,a county hospital. Applications of LC-MS based methods in toxicology and therapeutic drug monitoring in a hospital setting. I have a BS pharmaceutical sciences, MS in Chemistry and PhD from University of North Carolina at Greensboro, NC.
The current abstract is from our pilot study in human cellular metabolomics carried out in the Nano-engineering Dept in North Carolina A and T University, Greensboro, NC in my previous role before transition to clinical MS recently (the work was not present at any scientific conferences. The pilot study is to assess the cytotoxic effects of Silica Nanoparticles (SiNPs) on human lung epithelial cells (A549s) to identify early stage metabolite biomarkers relevant to nanomaterial toxicity. We employing HILIC model chromatography UPLC-HRMS nontargeted metabolomics workflow
Relevant Financial Disclosures
(within past 24 months)
No relevant financial relationship(s) to disclose.
Abstract
Non-Functionalized silica nanoparticles (SiNPs) are some of the widely used nanomaterial in diverse industrial sectors and nanoparticle based drug delivery applications. In industrial manufacturing environment SiNPs can possibly comes in contact with employees. Studies of cellular level toxicity effects of SiNPs in human cell lines are pivotal in the metabolite based biomarker discovery. Human lung epithelial cells (A549) are used to decipher the overall cellular level metabolic changes, non-targeted metabolite profiling with HILIC (hydrophilic interaction liquid chromatography) UPLC-HRMS method in a data dependent (DDA) mode was developed for this study. From the identified metabolome data and corresponding dysregulation in the metabolome of A549 biochemical pathways, our preliminary finding indicated 8 nm SiNPs elicit observable effects on the A549 cellular metabolism over larger SiNPs.The study identified some insights in early stage selective metabolite markers for nanomaterial related cytotoxicity in human cell line.Identified metabolites were annotated to pathways related to glutathione mediated detoxification, amino acid degradation, central carbohydrate metabolism and nucleotide metabolism with statistical significance (p < 0.01).
Introduction
Non-Functionalized amorphous silica nanoparticles(SiNPs) are used in nano material manufacturing and nanoparticle based drug formulation studies,they can possibly come in contact with humans via skin and inhalation routes. We have developed an untargeted UPLC HRMS method to assess cellular level metabolic dysregulation of SiNPs on human lung epithelial cells (A549).
Methods
A549 cell line control group and SiNPs (spherical 8, 80 and 120 nm dia.) as exposure groups were used in the metabolite profiling experiment. HILIC mode UPLC-HRMS positive and negative polarity methods were developed to acquire tandem MS data in a data dependent MS (DDA) mode.Pooled QC was used to monitor MS method and overall LC-MS hardware performance and method robustness. XCMS online (Scripps Research Institute) and Compound Discoverer 2.1 were used for metabolome MS data processing and statistical analysis.Retention time alignment based MS1 precursor peak areas were used in relative quantitation between experimental groups.The metabolite pathway annotation was carried out with KEGG and BioCyc pathway representation.
Results and conclusions
The 8 nm SiNPs tend to affect the cellular level physiology to a wider extend over larger SiNP exposure groups.Data analysis revealed significant changes in relative metabolome profiles, p-value < 0.01.Differential changes in metabolites were annotated to pathways of glutathione detoxification, glutathione redox reactions II, central carbon metabolism, amino acid degradation pathways and t-RNA re-charging.
Acknowledgements
Shyam Arvamudhan, PhD
North Carolina A and T University, Greensboro, NC.
Daniel Todd, PhD
Mass Spectrometry Facility
UNC Greensboro.
= Discovery stage.
= Translation stage.
= Clinically available.
