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Vasilis Vasiliou, James Jester, David Thompson, Georgia Charkoftaki; Untargeted metabolomics reveal lipid profile changes in rabbit cornea after sulfur mustard exposure. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1407.
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© ARVO (1962-2015); The Authors (2016-present)
Sulfur mustard (SM) is a highly reactive alkylating agent that exerts local cytotoxic effects on eyes. Ocular injuries can be followed by delayed irreversible corneal injuries. The aim of this study was to: (i) characterize the ex-vivo depth of injury (DoI) response of rabbit corneas exposed to mechlorethamine hydrochloride nitrogen mustard (NM), a sulfur mustard simulant, and (ii) use untargeted metabolomics to elucidate the mechanism of the NM-mediated ocular damage.
Intact ex vivo rabbit eyes were placed in serum-free DMEM organ culture. NM (0, 1, 2.5, 5 or 10 mg/ml) was applied to the central cornea for 10 min using a 5 mm filter disk. Corneas were then cultured for 3 or 24 h before being (i) fixed for DoI analysis (TUNEL and Alexa 488- labeled phalloidin) or (ii) snap frozen for metabolomic analysis (LC-MS/MS, Waters Xevo G2-XS QTof). A method detecting lipids (lipidomics) was developed for rabbit cornea and the samples were run in both positive and negative mode. Multivariate analysis was used to compare vehicle and NM-treated corneas.
No DoI damage was caused by 3 h NM exposure. At 24 h, 1 mg/ml NM caused erosion of the corneal epithelium, but no damage to the underlying stroma. Damage caused by 2.5 mg/ml NM extended to a 50% depth of the corneal stroma, while 5 and 10 mg/ml completely penetrated the corneal stroma. Metabolomics showed an altered lipid profile occurred in NM-treated corneas at 24 h exposure. All NM doses were shown to increase sphingomyelin (SP) levels 1.5-5 fold (p<0.1). Putative ions altered were C15-C18 and C26 SP. LC-MS/MS was able to detect changes at the lowest NM dose (1 mg). In most samples, there was a trend of increased ion abundance with increasing NM dose.
Doses of NM were identified that caused different DoIs to the cornea, from slight (1 mg/ml) to severe damage (5 and 10 mg/ml) extending down to the corneal endothelium. Metabolomics revealed alterations in sphingomyelins. These lipids have been linked to many vital corneal cell processes, such as apoptosis and cell growth. We propose that SP analysis provides a sensitive method for evaluating novel approaches to prevent SM-induced ocular damage.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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