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™.
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.
College of Pharmacy, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul, 120-808, Republic of Korea.
Seunghye
S
Choi
Miri
M
Lee
Su-Hyon
SH
Lee
Haeng-Sun
HS
Jung
Seol-Yeong
SY
Kim
Tae-Young
TY
Chung
Tae-boo
TB
Choe
Young-Jin
YJ
Chun
Kyung-Min
KM
Lim
D001548:Benzalkonium Compounds / Q000633:toxicity; D015415:Biomarkers / Q000378:metabolism; D002478:Cells, Cultured; D051766:Early Growth Response Protein 1 / Q000235:genetics*; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000473:pathology; D006801:Humans; D007509:Irritants / Q000633:toxicity; D008565:Membrane Proteins / Q000235:genetics*; D008954:Models, Biological; D017830:Octoxynol / Q000633:toxicity; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000378:metabolism; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate / Q000633:toxicity; D013501:Surface-Active Agents / Q000633:toxicity*; D015854:Up-Regulation / Q000187:drug effects
D001548:Benzalkonium Compounds; D015415:Biomarkers; C487795:CNFN protein, human; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D007509:Irritants; D008565:Membrane Proteins; D012333:RNA, Messenger; D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate; D017830:Octoxynol