Publications by authors named "James E Keating"

9 Publications

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Reactive Oxygen Species, Mitochondrial Membrane Potential, and Cellular Membrane Potential Are Predictors of E-Liquid Induced Cellular Toxicity.

Nicotine Tob Res 2020 12;22(Suppl 1):S4-S13

Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, NC.

Introduction: The use of flavors in electronic cigarettes appeals to adults and never-smoking youth. Consumption has rapidly increased over the last decade, and in the U.S. market alone, there are over 8000 unique flavors. The U.S. Food and Drug Administration (FDA) has begun to regulate e-liquids, but many have not been tested, and their impact, both at the cellular level, and on human health remains unclear.

Methods: We tested e-liquids on the human cell line HEK293T and measured toxicity, mitochondrial membrane potential (ΔΨ  m), reactive oxygen species production (ROS), and cellular membrane potential (Vm) using high-throughput screening (HTS) approaches. Our HTS efforts included single-dose and 16-point dose-response curves, which allowed testing of ≥90 commercially available e-liquids in parallel to provide a rapid assessment of cellular effects as a proof of concept for a fast, preliminary toxicity method. We also investigated the chemical composition of the flavors via gas chromatography-mass spectrometry.

Results: We found that e-liquids caused a decrease in ΔΨ  m and Vm and an increase in ROS production and toxicity in a dose-dependent fashion. In addition, the presence of five specific chemical components: vanillin, benzyl alcohol, acetoin, cinnamaldehyde, and methyl-cyclopentenolone, but not nicotine, were linked with the changes observed in the cellular traits studied.

Conclusion: Our data suggest that ΔΨ  m, ROS, Vm, and toxicity may be indicative of the extent of cell death upon e-liquid exposure. Further research on the effect of flavors should be prioritized to help policy makers such as the FDA to regulate e-liquid composition.

Implications: E-liquid cellular toxicity can be predicted using parameters amenable to HTS. Our data suggest that ΔΨ  m, ROS, Vm, and toxicity may be indicative of the extent of cell death upon e-liquid exposure, and this toxicity is linked to the chemical composition, that is, flavoring components. Further research on the effect of flavors should be prioritized to help policy makers such as the FDA to regulate e-liquid composition.
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http://dx.doi.org/10.1093/ntr/ntaa177DOI Listing
December 2020

Cigarillos Compromise the Mucosal Barrier and Protein Expression in Airway Epithelia.

Am J Respir Cell Mol Biol 2020 12;63(6):767-779

Department of Pathology and Laboratory Medicine.

Smoking remains a leading cause of preventable morbidity and mortality worldwide. Despite a downward trend in cigarette use, less-regulated tobacco products, such as cigarillos, which are often flavored to appeal to specific demographics, such as younger people, are becoming increasingly popular. Cigar/cigarillo smoking has been considered a safer alternative to cigarettes; however, the health risks associated with cigar in comparison with cigarette smoking are not well understood. To address this knowledge gap, we characterized the effects of multiple brands of cigarillos on the airway epithelium using and models. To analyze these effects, we assessed the cellular viability and integrity of smoke-exposed primary airway cell cultures. We also investigated the protein compositions of apical secretions from cigarillo-exposed airway epithelial cultures and BAL fluid of cigarillo-exposed mice through label-free quantitative proteomics and determined the chemical composition of smoke collected from the investigated cigarillo products. We found that cigarillo smoke exerts similar or greater effects than cigarette smoke in terms of reduced cell viability; altered protein levels, including those of innate immune proteins; induced oxidative-stress markers; and greater nicotine delivery to cells. The analysis of the chemical composition of the investigated cigarillo products revealed differences that might be linked to the differential effects of these products on cell viability and protein abundance profiles, which have been associated with a range of health risks in the context of airway biology. These findings contradict the assumption that cigarillos might be safer and less harmful than cigarettes. Instead, our results indicate that cigarillo smoke is associated with equal or greater health risks and the same or increased airway toxicity compared with cigarette smoke.
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http://dx.doi.org/10.1165/rcmb.2019-0085OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790145PMC
December 2020

Alkali Metal Cationization of Tumor-associated Antigen Peptides for Improved Dissociation and Measurement by Differential Ion Mobility-Mass Spectrometry.

J Proteome Res 2020 08 20;19(8):3176-3183. Epub 2020 Jul 20.

Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.

Tandem mass spectrometry (MS/MS) is a highly sensitive and selective method for the detection of tumor-associated peptide antigens. These short, nontryptic sequences may lack basic residues, resulting in the formation of predominantly [peptide + H] ions in electrospray. These singly charged ions tend to undergo inefficient dissociation, leading to issues in sequence determination. Addition of alkali metal salts to the electrospray solvent can drive the formation of [peptide + H + metal] ions that have enhanced dissociation characteristics relative to [peptide + H] ions. Both previously identified tumor-associated antigens and predicted neoantigen sequences were investigated. The previously reported rearrangement mechanism in MS/MS of sodium-cationized peptides is applied here to demonstrate complete C-terminal sequencing of tumor-associated peptide antigens. Differential ion mobility spectrometry (DIMS) is shown to selectively enrich [peptide + H + metal] species by filtering out singly charged interferences at relatively low field strengths, offsetting the decrease in signal intensity associated with the use of alkali metal cations.
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http://dx.doi.org/10.1021/acs.jproteome.0c00157DOI Listing
August 2020

Flavored e-liquids increase cytoplasmic Ca levels in airway epithelia.

Am J Physiol Lung Cell Mol Physiol 2020 02 6;318(2):L226-L241. Epub 2019 Nov 6.

Marsico Lung Institute, University of North Carolina, Chapel Hill, North Carolina.

E-cigarettes are noncombustible, electronic nicotine-delivery devices that aerosolize an e-liquid, i.e., nicotine, in a propylene glycol-vegetable glycerin vehicle that also contains flavors. While the effects of nicotine are relatively well understood, more information regarding the potential biological effects of the other e-liquid constituents is needed. This is a serious concern, because e-liquids are available in >7,000 distinct flavors. We previously demonstrated that many e-liquids affect cell growth/viability through an unknown mechanism. Since Ca is a ubiquitous second messenger that regulates cell growth, we characterized the effects of e-liquids on cellular Ca homeostasis. To better understand the extent of this effect, we screened e-liquids for their ability to alter cytosolic Ca levels and found that 42 of 100 flavored e-liquids elicited a cellular Ca response. Banana Pudding (BP) e-liquid, a representative e-liquid from this group, caused phospholipase C activation, endoplasmic reticulum (ER) Ca release, store-operated Ca entry (SOCE), and protein kinase C (PKCα) phosphorylation. However, longer exposures to BP e-liquid depleted ER Ca stores and inhibited SOCE, suggesting that this e-liquid may alter Ca homeostasis by short- and long-term mechanisms. Since dysregulation of Ca signaling can cause chronic inflammation, ER stress, and abnormal cell growth, flavored e-cigarette products that can elicit cell Ca responses should be further screened for potential toxicity.
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http://dx.doi.org/10.1152/ajplung.00123.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052665PMC
February 2020

Computational modeling and confirmation of leukemia-associated minor histocompatibility antigens.

Blood Adv 2018 08;2(16):2052-2062

Lineberger Comprehensive Cancer Center.

T-cell responses to minor histocompatibility antigens (mHAs) mediate both antitumor immunity (graft-versus-leukemia [GVL]) and graft-versus-host disease (GVHD) in allogeneic stem cell transplant. Identifying mHAs with high allele frequency, tight binding affinity to common HLA molecules, and narrow tissue restriction could enhance immunotherapy against leukemia. Genotyping and HLA allele data from 101 HLA-matched donor-recipient pairs (DRPs) were computationally analyzed to predict both class I and class II mHAs likely to induce either GVL or GVHD. Roughly twice as many mHAs were predicted in HLA-matched unrelated donor (MUD) stem cell transplantation (SCT) compared with HLA-matched related transplants, an expected result given greater genetic disparity in MUD SCT. Computational analysis predicted 14 of 18 previously identified mHAs, with 2 minor antigen mismatches not being contained in the patient cohort, 1 missed mHA resulting from a noncanonical translation of the peptide antigen, and 1 case of poor binding prediction. A predicted peptide epitope derived from GRK4, a protein expressed in acute myeloid leukemia and testis, was confirmed by targeted differential ion mobility spectrometry-tandem mass spectrometry. T cells specific to UNC-GRK4-V were identified by tetramer analysis both in DRPs where a minor antigen mismatch was predicted and in DRPs where the donor contained the allele encoding UNC-GRK4-V, suggesting that this antigen could be both an mHA and a cancer-testis antigen. Computational analysis of genomic and transcriptomic data can reliably predict leukemia-associated mHA and can be used to guide targeted mHA discovery.
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http://dx.doi.org/10.1182/bloodadvances.2018022475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113610PMC
August 2018

Dual Emitter Nano-Electrospray Ionization Coupled to Differential Ion Mobility Spectrometry-Mass Spectrometry for Shotgun Lipidomics.

Anal Chem 2018 08 23;90(15):9117-9124. Epub 2018 Jul 23.

Department of Chemistry , The University of North Carolina at Chapel Hill , Chapel Hill , North Carolina 27599 United States.

Current lipidomics workflows are centered around acquisition of large data sets followed by lengthy data processing. A dual nESI-DIMS-MS platform was developed to perform real-time relative quantification between samples, providing data required for biomarker discovery and validation more quickly than traditional ESI-MS approaches. Nanosprayer activity and DIMS compensation field settings were controlled by a LabVIEW program synced to the accumulation portion of the ion trap scan function, allowing for full integration of the platform with a commercial mass spectrometer. By comparing samples with short electrospray pulses rather than constant electrospray, the DIMS and MS performance is normalized within an experiment, as signals are compared between individual mass spectra (ms time scale) rather than individual experiments (min-hr time scale). The platform was validated with lipid standards and extracts from nitrogen-deprived microalgae. Dual nESI-DIMS requires minimal system modification and is compatible with all traditional ion activation techniques and mass analyzers, making it a versatile improvement to shotgun lipidomics workflows.
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http://dx.doi.org/10.1021/acs.analchem.8b01528DOI Listing
August 2018

Evaluation of e-liquid toxicity using an open-source high-throughput screening assay.

PLoS Biol 2018 03 27;16(3):e2003904. Epub 2018 Mar 27.

Marsico Lung Institute/Cystic Fibrosis Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

The e-liquids used in electronic cigarettes (E-cigs) consist of propylene glycol (PG), vegetable glycerin (VG), nicotine, and chemical additives for flavoring. There are currently over 7,700 e-liquid flavors available, and while some have been tested for toxicity in the laboratory, most have not. Here, we developed a 3-phase, 384-well, plate-based, high-throughput screening (HTS) assay to rapidly triage and validate the toxicity of multiple e-liquids. Our data demonstrated that the PG/VG vehicle adversely affected cell viability and that a large number of e-liquids were more toxic than PG/VG. We also performed gas chromatography-mass spectrometry (GC-MS) analysis on all tested e-liquids. Subsequent nonmetric multidimensional scaling (NMDS) analysis revealed that e-liquids are an extremely heterogeneous group. Furthermore, these data indicated that (i) the more chemicals contained in an e-liquid, the more toxic it was likely to be and (ii) the presence of vanillin was associated with higher toxicity values. Further analysis of common constituents by electron ionization revealed that the concentration of cinnamaldehyde and vanillin, but not triacetin, correlated with toxicity. We have also developed a publicly available searchable website (www.eliquidinfo.org). Given the large numbers of available e-liquids, this website will serve as a resource to facilitate dissemination of this information. Our data suggest that an HTS approach to evaluate the toxicity of multiple e-liquids is feasible. Such an approach may serve as a roadmap to enable bodies such as the Food and Drug Administration (FDA) to better regulate e-liquid composition.
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http://dx.doi.org/10.1371/journal.pbio.2003904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5870948PMC
March 2018

Paper Spray Mass Spectrometry for High-Throughput Quantification of Nicotine and Cotinine.

Anal Methods 2018 28;10(1):46-50. Epub 2017 Nov 28.

Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 USA.

The rapid release of new tobacco products requires high-throughput quantitative methods to support tobacco research. Sample preparation for LC-MS and GC-MS is time consuming and limits throughput. Paper spray tandem mass spectrometry (PS-MS/MS) is proposed and validated as a simple and rapid method for quantification of nicotine and cotinine in complex matrices to support tobacco-related research. Air liquid interface (ALI) human tracheobronchial epithelial cell (HTBEC) cultures were exposed to tobacco smoke using a Vitrocell VC-10 smoking machine. Apical culture washes (phosphate buffered saline, PBS) and basolateral media were analyzed with the PS-MS/MS method. GC-MS/MS was used as a comparative quantitative technique. The PS-MS/MS approach allowed for direct spotting of samples on the paper substrate, whereas the GC-MS/MS method required additional sample preparation in the form of solvent-solvent extraction. Limits of quantitation (LOQs) were higher with the PS-MS/MS approach than GC-MS/MS, but still below the relevant concentrations found in HTBEC smoke exposure experiments as well as most clinical applications. PS-MS/MS is readily achieved on mass spectrometers that include atmospheric pressure inlets, and allows for convenient quantification from complex matrices that would otherwise require additional sample preparation and chromatographic separation.
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http://dx.doi.org/10.1039/C7AY02204BDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5858713PMC
November 2017

Flavored e-cigarette liquids and cinnamaldehyde impair respiratory innate immune cell function.

Am J Physiol Lung Cell Mol Physiol 2017 08 11;313(2):L278-L292. Epub 2017 May 11.

Curriculum in Toxicology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina;

Innate immune cells of the respiratory tract are the first line of defense against pathogenic and environmental insults. Failure of these cells to perform their immune functions leaves the host susceptible to infection and may contribute to impaired resolution of inflammation. While combustible tobacco cigarettes have been shown to suppress respiratory immune cell function, the effects of flavored electronic cigarette liquids (e-liquids) and individual flavoring agents on respiratory immune cell responses are unknown. We investigated the effects of seven flavored nicotine-free e-liquids on primary human alveolar macrophages, neutrophils, and natural killer (NK) cells. Cells were challenged with a range of e-liquid dilutions and assayed for their functional responses to pathogenic stimuli. End points included phagocytic capacity (neutrophils and macrophages), neutrophil extracellular trap formation, proinflammatory cytokine production, and cell-mediated cytotoxic response (NK cells). E-liquids were then analyzed via mass spectrometry to identify individual flavoring components. Three cinnamaldehyde-containing e-liquids exhibited dose-dependent broadly immunosuppressive effects. Quantitative mass spectrometry was used to determine concentrations of cinnamaldehyde in each of the three e-liquids, and cells were subsequently challenged with a range of cinnamaldehyde concentrations. Cinnamaldehyde alone recapitulated the impaired function observed with e-liquid exposures, and cinnamaldehyde-induced suppression of macrophage phagocytosis was reversed by addition of the small-molecule reducing agent 1,4-dithiothreitol. We conclude that cinnamaldehyde has the potential to impair respiratory immune cell function, illustrating an immediate need for further toxicological evaluation of chemical flavoring agents to inform regulation governing their use in e-liquid formulations.
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http://dx.doi.org/10.1152/ajplung.00452.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5582929PMC
August 2017