title: Cytotoxic effect and possible mechanisms of Tetracaine on human corneal epithelial cells in vitro.
abstract: OBJECTIVE
  To demonstrate the cytotoxic effect and possible mechanisms of Tetracaine on human corneal epithelial (HCEP) cells in vitro.
  
  
  METHODS
  In vitro cultured HCEP cell were treated with Tetracaine hydrochloride at different doses for different times, and their morphology, viability, and plasma membrane permeability were detected by light microscopy, methyl thiazolyl tetrazolium (MTT) assay, and acridine orange (AO)/ethidium bromide (EB) staining, respectively. Their cell cycle progression, phosphatidylserine orientation in plasma membrane, and mitochondrial membrane potential (MTP) were assessed by flow cytometry. DNA fragmentation, ultrastructure, caspase activation, and the cytoplasmic apoptosis inducing factor (AIF) and cytochrome c (Cyt. c) along with the expression of B-cell lymphoma-2 (Bcl-2) family proteins were examined by gel electrophoresis, transmission electron microscope, enzyme linked immunosorbent assay (ELISA), and Western blot, respectively.
  
  
  RESULTS
  After exposed to Tetracaine at doses from 10.0 to 0.3125 g/L, the HCEP cells showed dose- and time-dependent morphological abnormality and typical cytopathic effect, viability decline, and plasma membrane permeability elevation. Tetracaine induced phosphatidylserine externalization, DNA fragmentation, G1 phase arrest, and ultrastructural abnormality and apoptotic body formation. Furthermore, Tetracaine at a dose of 0.3125 g/L also induced caspase-3, -9 and -8 activation, MTP disruption, up-regulation of the cytoplasmic amount of Cyt. c and AIF, the expressions of Bax and Bad, and down-regulation of the expressions of Bcl-2 and Bcl-xL.
  
  
  CONCLUSIONS
  Tetracaine above 0.3125 g/L (1/32 of its clinical applied dosage) has a dose- and time-dependent cytotoxicity to HCEP cells in vitro, with inducing cell apoptosis via a death receptor-mediated mitochondrion-dependent pathway.
authors.affiliation: Key Laboratory for Corneal Tissue Engineering, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, Shandong Province, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Pang
authors.affiliation: Key Laboratory for Corneal Tissue Engineering, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, Shandong Province, China.
authors.forename: Ting-Jun
authors.initials: TJ
authors.lastname: Fan
keywords: Tetracaine; apoptosis; cytotoxicity; human cornel epithelial cells; mitochondrion