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. 2018 May 21;13(5):e0196735.
doi: 10.1371/journal.pone.0196735. eCollection 2018.

Exploration of alternative test methods to evaluate phototoxicity of ophthalmic agents by using Statens Seruminstitut Rabbit Cornea cell lines and 3D human reconstituted cornea models

Soyoung Kim  1   2 , Ki Hwan Choi  1 , Jaesuk Yun  2
Affiliations

Exploration of alternative test methods to evaluate phototoxicity of ophthalmic agents by using Statens Seruminstitut Rabbit Cornea cell lines and 3D human reconstituted cornea models

Soyoung Kim et al. PLoS One. .

Abstract

Many chemicals have been reported to induce phototoxicity. The absorbance of light energy within the sunlight range is a common characteristic of phototoxicity. The 3T3 NRU phototoxicity test (PT) in 3T3 mouse skin fibroblasts has been used to identify the phototoxic potential induced by excited chemicals after exposure to ultra violet (UV). However, as phototoxicity may occur in ocular cells, it is necessary to develop a more suitable test for cornea-derived cells. In this study, we attempted to establish a new in vitro PT method in rabbit corneal cell lines (SIRC). We evaluated five ophthalmic agents, ciprofloxacin, levofloxacin, lomefloxacin, norfloxacin, and tetracycline, for their cytotoxic potential and in vitro phototoxicity. The results obtained using 3D human corneal models revealed that the UV-induced eye tissue toxicity by the test substances showed good correlation with those obtained using the in vitro phototoxicity test. However, the results from the 3D PT for ciprofloxacin, norfloxacin, and tetracycline in the 3D human cornea model were only partially comparable. Therefore, we suggest the SIRC cell line as a new phototoxicity test model; however, a sequential testing strategy, such as 3D PT, was also proposed to obtain relevant information for topical eye agents.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Spectral properties of chlorpromazine (positive control), ciprofloxacin, levofloxacin, lomefloxacin, norfloxacin, and tetracycline (test substances), and L-histidine and sodium lauryl sulfate (negative controls).
Fig 2
Fig 2. Irradiation sensitivity of SIRC cells.
Cell viability showed a dose-dependent response to irradiation, with 85.2%±4.9% after irradiation at 5 J/cm2, 73.8%±3.4% after irradiation at 10 J/cm2, and 66.4%±2.9% after irradiation at 20 J/cm2. The data are expressed as the mean ± S.E. (n = 5).
Fig 3
Fig 3
Phototoxicity evaluation of the ophthalmic agents in SIRC cells: A) chlorpromazine (positive control), B) ciprofloxacin, C) levofloxacin, D) lomefloxacin, E) norfloxacin, F) tetracycline, G) L-histidine (negative control), and H) sodium lauryl sulfate (negative control). The closed circle and open circle represent data from the nonirradiated groups and UV-irradiated groups, respectively. Phototoxic chemicals induced dose-response curve shift with UV-irradiation. Fitting of the curve to the data was performed by a non-linear regression method (n = 6).
Fig 4
Fig 4
Hematoxylin and eosin staining of 3D human cornea models (HCE) of the ophthalmic agent, with and without UV irradiation of 6 J/cm2: A) chlorpromazine (positive control), B) ciprofloxacin, C) norfloxacin, D) tetracycline, E) L-histidine (negative control), and F) sodium lauryl sulfate (negative control).
See this image and copyright information in PMC

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