title: Mechanisms underlying benzyl alcohol cytotoxicity (triamcinolone acetonide preservative) in human retinal pigment epithelial cells.
abstract: OBJECTIVE
  Benzyl alcohol (BA) is the preservative in triamcinolone acetonide (TA) suspensions, which are used in treating vitreoretinal diseases and during surgery. This paper investigates the molecular mechanisms and signaling pathways underlying BA toxicity in human retinal pigment epithelial (RPE) cells.
  
  
  METHODS
  Cultured human RPE cells from the ARPE-19 cell line were exposed to culture medium alone (control) or with BA (0.0225, 0.225, 0.9, 3, or 9 mg/mL) for up to 6 hours. BA toxicity was assessed by TUNEL assay, propidium iodide/annexin V-FITC staining and flow cytometry, caspase activation assay, caspase and apoptosis inhibition assays, mitochondrial transmembrane potential by rhodamine staining and flow cytometry, reactive oxygen species by chemiluminescence, and apoptosis-inducing factor staining.
  
  
  RESULTS
  BA caused RPE cell death not only by necrosis but also by apoptosis, evidenced by exposure to 9 mg/mL BA for 6 hours leading to 19.0% early apoptotic cells and 64.2% apoptotic necrotic cells. Apoptotic signaling involved the immediate production of reactive oxygen species, activation of caspase-8, impairment of the mitochondrial transmembrane potential, and further activation of caspase-9 and -3. In addition, BA induced translocation of apoptosis-inducing factor into the nucleus, indicating caspase-independent apoptosis.
  
  
  CONCLUSIONS
  BA leads to necrosis of RPE cells and triggers mitochondrial apoptosis through both caspase-dependent and - independent pathways. Extreme caution is suggested in the intraocular use of TA suspensions and meticulous evaluation before adoption of BA as a preservative in the future development of ophthalmic formulations.
authors.affiliation: Department of Opthalmology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
authors.forename: Yi-Sheng
authors.initials: YS
authors.lastname: Chang
authors.forename: Chiou-Feng
authors.initials: CF
authors.lastname: Lin
authors.forename: Chao-Liang
authors.initials: CL
authors.lastname: Wu
authors.forename: Pao-Ying
authors.initials: PY
authors.lastname: Kuo
authors.forename: Fong-Sen
authors.initials: FS
authors.lastname: Wu
authors.forename: Chi-Chang
authors.initials: CC
authors.lastname: Shieh
authors.forename: Po-Wu
authors.initials: PW
authors.lastname: Gean
authors.forename: Shur-Tzu
authors.initials: ST
authors.lastname: Chen
authors.forename: Muh-Shy
authors.initials: MS
authors.lastname: Chen
authors.forename: Wen-Chuan
authors.initials: WC
authors.lastname: Wu
authors.forename: Ming-Hong
authors.initials: MH
authors.lastname: Tai
authors.forename: Sung-Huei
authors.initials: SH
authors.lastname: Tseng
mesh_terms: D017209:Apoptosis; D051033:Apoptosis Inducing Factor / Q000378:metabolism; D019905:Benzyl Alcohol / Q000008:administration & dosage / Q000633:toxicity*; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004334:Drug Administration Schedule; D004789:Enzyme Activation; D005938:Glucocorticoids; D006801:Humans; D008564:Membrane Potentials; D008928:Mitochondria; D009336:Necrosis; D011310:Preservatives, Pharmaceutical / Q000008:administration & dosage / Q000633:toxicity*; D017382:Reactive Oxygen Species / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D015398:Signal Transduction; D013194:Staining and Labeling; D013997:Time Factors; D014018:Tissue Distribution / Q000187:drug effects; D014222:Triamcinolone Acetonide
chemical_list: D051033:Apoptosis Inducing Factor; D005938:Glucocorticoids; D011310:Preservatives, Pharmaceutical; D017382:Reactive Oxygen Species; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D014222:Triamcinolone Acetonide; D019905:Benzyl Alcohol