July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Glutathione Supplementation May Enhance Buthionine Sulfoximine-Induced Cell Death in Immortalized Human Meibomian Gland Epithelial Cells
Author Affiliations & Notes
  • William Ngo
    The University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Jason J Nichols
    The University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Footnotes
    Commercial Relationships   William Ngo, None; Jason Nichols, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 284. doi:
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    • Get Citation

      William Ngo, Jason J Nichols; Glutathione Supplementation May Enhance Buthionine Sulfoximine-Induced Cell Death in Immortalized Human Meibomian Gland Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):284.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Oxidative damage is modeled in various cell culture models by using buthionine sulfoximine (BSO) to block glutathione synthesis via γ-glutamylcysteine synthetase inhibition. Without glutathione to neutralize radical species, oxidative damage accumulates and results in cell death. To develop a human meibomian gland oxidative damage cell culture model, BSO have been shown to reduce HMGEC cell viability, however the relationship between BSO and glutathione has not been studied in HMGECs. The purpose of this study was to determine the effect of glutathione monoethyl ester (GME) supplementation against BSO-induced cell death.

Methods : HMGECs (Schepens Eye Research Institute, MA, USA) were grown to confluence in keratinocyte serum free media (KSFM) supplemented with 5ng/mL epithelial growth factor (EGF) and 5µg/mL bovine pituitary extract (BPE). The BSO was prepared by dissolving BSO in PBS and adjusting pH to 7.0. Glutathione was prepared by dissolving GME in PBS. Cells were seeded at a density of 135,000/well into 24-well plates containing KSFM and allowed to rest overnight. Cells were treated with equal parts Dulbecco’s Modified Eagle Medium and Ham’s F12, supplemented with EGF and 10% fetal bovine serum, along with the following experimental conditions: a) PBS (control), b) 50mM BSO, c) 50mM BSO+4mM GME, and d) 4mM GME only. Each condition was performed in technical triplicates and the entire experiment was duplicated. Cell viability was measured using a methylthiazolyldiphenyl tetrazolium (MTT, 0.5mg/mL) assay at t=0hr (no exposure to BSO), and t=24hr. The difference in cell viability among the conditions was tested using a 2-way ANOVA.

Results : At t=0hr, there was no significant difference in cell viability across all conditions compared to the PBS control (all p>0.62). At t=24hr, mean cell viability was 73%, 46%, and 58% relative to the PBS control for the 50mM BSO, 50mM BSO + 4mM GME, and 4mM GME conditions, respectively (all p<0.01). While there was no significant difference in cell viability between 50mM BSO and 4mM GME conditions (p=0.16), the combined 50mM BSO+4mM GME reduced cell viability to a greater extent than just 50mM BSO alone (p=0.01).

Conclusions : The supplementation of GME appeared to enhance BSO cytotoxicity in HMGECs, which was unexpected given the mechanism of BSO. Further experiments are required to verify the mechanism of cell death.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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