Ethanol treatment induces significant cell death in porcine corneal fibroblasts.
OBJECTIVE
To explore the effect of ethanol treatment on corneal stromal cells.


METHODS
Primary porcine corneal fibroblasts from passages 3 to 5 were treated with ethanol at concentrations of 10%, 15%, 20%, and 50% for 30 seconds. A control group was treated with phosphate-buffered saline (PBS) for 30 seconds. Morphologic changes were documented with phase-contrast microscopy, and the growth curves were examined with a PicoGreen assay. Cellular viability was examined with an ethidium homodimer and calcein-AM stain, whereas cellular apoptosis and/or necrosis were analyzed by a YO-PRO-1 dye/propidium iodide apoptosis assay coupled with flow cytometry and further confirmed with a genomic DNA pattern assay. Cellular toxicity was examined with a lactate dehydrogenase (LDH) assay.


RESULTS
Significant cell rounding and detachment from the culture dish were noticed after 20% ethanol treatment of 30 seconds, despite that the cell morphology remained unchanged in the PBS and 10% and 15% ethanol groups. Twenty percent ethanol induced significant cellular toxicity, causing cell death as shown by ethidium homodimer and calcein-AM stain, YO-PRO-1 dye/propidium iodide apoptosis assay, and LDH assay, although 10% and 15% ethanol caused minimal changes to corneal fibroblasts. Cellular death was most significant 6 hours after the 20% ethanol treatment. The genomic DNA pattern revealed intact DNA in the control, 10% ethanol, and 15% ethanol groups at all times, whereas DNA smearing was noticed at 48 hours after the 20% ethanol treatment. However, none of the DNA examined revealed significant DNA laddering patterns of apoptosis. Fifty percent ethanol treatment of 30 seconds resulted in cell fixation and cell death.


CONCLUSIONS
Treatment with 20% ethanol for 30 seconds induced significant porcine corneal fibroblast cell death, whereas 10% and 15% ethanol treatment of 30 seconds caused minimal changes. We propose that, when applied for 30 seconds, 20% ethanol is the threshold level that causes cell death in cultured porcine corneal fibroblasts.
Chang
Shu-Wen
SW
Department of Ophthalmology and Medical Research, Far Eastern Memorial Hospital, Ban-Chiao, Taiwan. swchang@mail.femh.org.tw
Chou
San-Fang
SF
Wang
Yao-Horng
YH
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D018929:Cell Culture Techniques; D002470:Cell Survival; D003319:Corneal Stroma / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D053938:DNA Fragmentation / Q000187:drug effects*; D000431:Ethanol / Q000633:toxicity*; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D005434:Flow Cytometry; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D012997:Solvents / Q000633:toxicity*; D013552:Swine
D012997:Solvents; D000431:Ethanol; D007770:L-Lactate Dehydrogenase