title: Benzalkonium chloride suppresses rabbit corneal endothelium intercellular gap junction communication.
abstract: OBJECTIVE
  Gap junction intercellular communication (GJIC) plays a critical role in the maintenance of corneal endothelium homeostasis. We determined if benzalkonium chloride (BAK) alters GJIC activity in the rabbit corneal endothelium since it is commonly used as a drug preservative in ocular eyedrop preparations even though it can have cytotoxic effects.
  
  
  METHODS
  Thirty-six adult New Zealand albino rabbits were randomly divided into three groups. BAK at 0.01%, 0.05%, and 0.1% was applied twice daily to one eye of each of the rabbits in one of the three groups for seven days. The contralateral untreated eyes were used as controls. Corneal endothelial morphological features were observed by in vivo confocal microscopy (IVCM). Immunofluorescent staining resolved changes in gap junction integrity and localization. Western blot analysis and RT-PCR evaluated changes in levels of connexin43 (Cx43) and tight junction zonula occludens-1 (ZO-1) gene and protein expression, respectively. Cx43 and ZO-1 physical interaction was detected by immunoprecipitation (IP). Primary rabbit corneal endothelial cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing BAK for 24 hours. The scrape-loading dye transfer technique (SLDT) was used to assess GJIC activity.
  
  
  RESULTS
  Topical administration of BAK (0.05%, 0.1%) dose dependently disrupted corneal endothelial cell morphology, altered Cx43 and ZO-1 distribution and reduced Cx43 expression. BAK also markedly induced increases in Cx43 phosphorylation status concomitant with decreases in the Cx43-ZO-1 protein-protein interaction. These changes were associated with marked declines in GJIC activity.
  
  
  CONCLUSIONS
  The dose dependent declines in rabbit corneal endothelial GJIC activity induced by BAK are associated with less Cx43-ZO-1 interaction possibly arising from increases in Cx43 phosphorylation and declines in its protein expression. These novel changes provide additional evidence that BAK containing eyedrop preparations should be used with caution to avoid declines in corneal transparency resulting from losses in GJIC activity and endothelial function.
authors.affiliation: Eye Institute and affiliated Xiamen Eye Center of Xiamen University; Fujian Provincial Key Laboratory of Ophthalmology and Vision Science, Xiamen, Fujian, China.
authors.forename: Zhenhao
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Institute of Stem Cell and Regenerative Medicine, Medical College, Xiamen University, Xiamen, Fujian, China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Eye Institute and affiliated Xiamen Eye Center of Xiamen University; Fujian Provincial Key Laboratory of Ophthalmology and Vision Science, Xiamen, Fujian, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Xie
authors.affiliation: Eye Institute and affiliated Xiamen Eye Center of Xiamen University; Fujian Provincial Key Laboratory of Ophthalmology and Vision Science, Xiamen, Fujian, China.
authors.forename: Juxin
authors.initials: J
authors.lastname: Pan
authors.affiliation: Eye Institute and affiliated Xiamen Eye Center of Xiamen University; Fujian Provincial Key Laboratory of Ophthalmology and Vision Science, Xiamen, Fujian, China.
authors.forename: Fanfei
authors.initials: F
authors.lastname: Liu
authors.affiliation: Eye Institute and affiliated Xiamen Eye Center of Xiamen University; Fujian Provincial Key Laboratory of Ophthalmology and Vision Science, Xiamen, Fujian, China.
authors.forename: Xuezhi
authors.initials: X
authors.lastname: Li
authors.affiliation: Eye Institute and affiliated Xiamen Eye Center of Xiamen University; Fujian Provincial Key Laboratory of Ophthalmology and Vision Science, Xiamen, Fujian, China.
authors.forename: Wensheng
authors.initials: W
authors.lastname: Chen
authors.affiliation: Eye Institute and affiliated Xiamen Eye Center of Xiamen University; Fujian Provincial Key Laboratory of Ophthalmology and Vision Science, Xiamen, Fujian, China.
authors.forename: Jiaoyue
authors.initials: J
authors.lastname: Hu
authors.affiliation: Eye Institute and affiliated Xiamen Eye Center of Xiamen University; Fujian Provincial Key Laboratory of Ophthalmology and Vision Science, Xiamen, Fujian, China.
authors.forename: Zuguo
authors.initials: Z
authors.lastname: Liu
mesh_terms: D000818:Animals; D001548:Benzalkonium Compounds / Q000633:toxicity*; D002450:Cell Communication / Q000187:drug effects*; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D017629:Gap Junctions / Q000187:drug effects*; D015870:Gene Expression; D010766:Phosphorylation / Q000187:drug effects; D011310:Preservatives, Pharmaceutical / Q000633:toxicity*; D061251:Primary Cell Culture; D011817:Rabbits; D062826:Zonula Occludens-1 Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D001548:Benzalkonium Compounds; D018031:Connexin 43; D011310:Preservatives, Pharmaceutical; D062826:Zonula Occludens-1 Protein