Identification of cornifelin and early growth response-1 gene as novel biomarkers for in vitro eye irritation using a 3D reconstructed human cornea model MCTT HCE™
- 478 Downloads
Evaluation of the eye irritation is essential in the development of new cosmetic products. Draize rabbit eye irritation test has been widely used in which chemicals are directly applied to rabbit eye, and the symptoms and signs of eyes are scored. However, due to the invasive procedure, it causes substantial pain and discomfort to animals. Recently, we reported in vitro eye irritation test method using a 3D human corneal epithelial model (MCTT HCE™) which is reconstructed from remaining human tissues after a corneal transplantation. This model exhibited an excellent predictive capacity for 25 reference chemicals (sensitivity 100 %, specificity 77 % and accuracy 88 % vs. GHS). To improve the test performance, we explored new biomarkers for the eye irritation through transcriptomic approach. Three surfactants were selected as model eye irritants that include sodium lauryl sulfate, benzalkonium chloride and triton X-100. After test chemicals were treated, we investigated differentially expressed genes through a whole-gene microarray (Affymetrix GeneChip® Human Gene 2.0 ST Array, 48,000 probes). As a result, we identified that mRNAs of cornifelin (CNFN), a constituent of the insoluble cornified cell envelope of stratified squamous epithelia, and early growth response-1 (EGR1), a nuclear transcriptional regulator, were significantly up-regulated by all three irritants. Up-regulation of CNFN and EGR1 was further confirmed by Q-RT-PCR, and immunohistochemistry revealed increased level of CNFN in irritant-treated tissues, supporting the relevance of CNFN and EGR1 as new biomarkers for eye irritation.
KeywordsEye irritation Alternative to animal test methods Transcriptomics Cornifelin Early growth response-1 gene
This work is supported by Ministry of Food and Drug Safety of Korea, Grant No. 13172MFDS987 and the National Research Foundation (NRF) Grant of the Korea government (MEST), No. NRF-2013R1A1A1007072.
- Adriaens E, Barroso J, Eskes C et al (2014) Retrospective analysis of the Draize test for serious eye damage/eye irritation: importance of understanding the in vivo endpoints under UN GHS/EU CLP for the development and evaluation of in vitro test methods. Arch Toxicol 88(3):701–723. doi: 10.1007/s00204-013-1156-8 CrossRefPubMedPubMedCentralGoogle Scholar
- Cotovio J, Grandidier M-H, Lelièvre D et al (2007) The use of the reconstructed human corneal model (HCE) to assess in vitro eye irritancy of chemicals. ALTeX 14:343–350Google Scholar
- Cotovio J, Grandidier MH, Lelievre D et al (2010) In vitro assessment of eye irritancy using the reconstructed human corneal Epithelial SkinEthic HCE model: application to 435 substances from consumer products industry. Toxicol in vitro 24(2):523–537. doi: 10.1016/j.tiv.2009.11.010 CrossRefPubMedGoogle Scholar
- Doucet O, Lanvin M, Thillou C et al (2006) Reconstituted human corneal epithelium: a new alternative to the Draize eye test for the assessment of the eye irritation potential of chemicals and cosmetic products. Toxicol in vitro 20(4):499–512. doi: 10.1016/j.tiv.2005.09.005 CrossRefPubMedGoogle Scholar
- Kaluzhny Y, Kandarova H, Hayden P, Kubilus J, d’Argembeau-Thornton L, Klausner M (2011) Development of the EpiOcular™ eye irritation test for hazard identification and labelling of eye irritating chemicals in response to the requirements of the EU cosmetics directive and REACH legislation. ATLA 39(4):339–364PubMedGoogle Scholar
- Liang H, Pauly A, Riancho L, Baudouin C, Brignole-Baudouin F (2011) Toxicological evaluation of preservative-containing and preservative-free topical prostaglandin analogues on a three-dimensional-reconstituted corneal epithelium system. Br J Ophthalmol 95(6):869–875. doi: 10.1136/bjo.2010.189449 CrossRefPubMedPubMedCentralGoogle Scholar
- Pauly A, Meloni M, Brignole-Baudouin F, Warnet JM, Baudouin C (2009) Multiple endpoint analysis of the 3D-reconstituted corneal epithelium after treatment with benzalkonium chloride: early detection of toxic damage. Invest Ophthalmol Vis Sci 50(4):1644–1652. doi: 10.1167/iovs.08-2992 CrossRefPubMedGoogle Scholar