title: Occludin gene expression as an early in vitro sign for mild eye irritation assessment.
abstract: OBJECTIVE
  To test a new multiple endpoint analysis (MEA) including occludin gene expression for screening the ocular irritation potential of tear substitutes on human corneal epithelium (HCE), an in vitro model proposed to limit the use of animal testing in pre-clinical studies.
  
  
  METHODS
  Four chemically-preserved and two non chemically-preserved tear substitutes were tested after acute (24h, 24h+24h post incubation) and repeated applications (for 72h) and compared to the positive control, benzalkonium chloride (BAK) at 0.1% and 0.01%, by assessing complementary parameters. Cellular viability was evaluated using MTT, histomorphologic analysis was performed on H&E stained vertical sections, IL-8 release was measured by ELISA, and occludin gene expression was quantified using qRT-PCR.
  
  
  RESULTS
  Cellular viability was moderately reduced by Perborate and Polyquad-preserved tear substitutes and dramatically reduced by BAK and by Thiomersal and Oxyd preserved tear substitutes. Thiomersal also increased IL-8 release. Occludin expression profiles were modified by the four chemically-preserved tear substitutes and by the mechanically-preserved Comod, but not by the mechanically-preserved Abak. The behavior of BAK and tear substitutes led us to propose a prediction model for the classification of different levels of irritants, mainly based on the occludin transcriptional study.
  
  
  CONCLUSIONS
  The versatility and sensitivity of the HCE model allowed the modeling of cumulative effects that may approach conditions obtained after long term application of tear substitutes. Thus, the modified MEA proposed in this study represents a valuable tool for in vitro eye irritation assessment with the power to detect mild irritants and subclinical eye irritant potential.
authors.affiliation: VitroScreen, In Vitro Research Laboratories, Milano, Italy. marisa.meloni@vitroscreen.com
authors.forename: Marisa
authors.initials: M
authors.lastname: Meloni
authors.forename: Aude
authors.initials: A
authors.lastname: Pauly
authors.forename: Barbara De
authors.initials: BD
authors.lastname: Servi
authors.forename: Béatrice Le
authors.initials: BL
authors.lastname: Varlet
authors.forename: Christophe
authors.initials: C
authors.lastname: Baudouin
mesh_terms: D002470:Cell Survival / Q000187:drug effects; D004396:Coloring Agents; D004353:Drug Evaluation, Preclinical; D004797:Enzyme-Linked Immunosorbent Assay; D005128:Eye Diseases / Q000139:chemically induced* / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000187:drug effects*; D005819:Genetic Markers; D006801:Humans; D066298:In Vitro Techniques; D016209:Interleukin-8 / Q000378:metabolism; D007509:Irritants / Q000633:toxicity*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D062793:Occludin; D009883:Ophthalmic Solutions / Q000633:toxicity; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013778:Tetrazolium Salts; D013844:Thiazoles
chemical_list: D004396:Coloring Agents; D005819:Genetic Markers; D016209:Interleukin-8; D007509:Irritants; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D009883:Ophthalmic Solutions; D012333:RNA, Messenger; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue