Effect of Benzalkonium, Mitomycin-C and Dexamethasone on Stress in Trabecular Meshwork Cells

Byung Moon Kim; Jae Woo Kim

doi:10.3341/jkos.2015.56.1.104

Journal List > J Korean Ophthalmol Soc > v.56(1) > 1010023

Go to TopGo to Top Go to BottomGo to Bottom

TOOLS

Kim and Kim: Effect of Benzalkonium, Mitomycin-C and Dexamethasone on Stress in Trabecular Meshwork Cells

Original Article

J Korean Ophthalmol Soc 2015;56(1):104-108.

Published online: 6 September 2015

DOI: https://doi.org/10.3341/jkos.2015.56.1.104

Effect of Benzalkonium, Mitomycin-C and Dexamethasone on Stress in Trabecular Meshwork Cells

Byung Moon Kim, MD, Jae Woo Kim, MD, PhD

Department of Ophthalmology, Catholic University of Daegu School of Medicine, Daegu, Korea

Address reprint requests to Jae Woo Kim, MD, PhD, Department of Ophthalmology, Daegu Catholic University Medical Center, #33 Duryugongwon-ro 17-gil, Nam-gu, Daegu 705-718, Korea, Tel: 82-53-650-4728, Fax: 82-53-627-0133 E-mail: jwkim@cu.ac.kr

Received 14 March 201419 May 2014 Accepted 19 December 2014

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Purpose

To investigate the effects of benzalkonium chloride (BAC), mitomycin C (MMC) and dexamethasone (DEX) on cellular stress in cultured human trabecular meshwork cell (HTMC) monolayers.

Methods

HTMCs were cultured in the inner Transwell chamber until confluence and then were exposed to BAC, MMC or DEX for 6 hours. The carboxyfluorescein permeability through the HTMC monolayer was measured using a spectrofluorometer at 532 nm after 2 hours in the outer chamber. The 3-[4, 5 -dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay was used to evaluate cellular viabilities.

Results

The carboxyfluorescein permeability through the HTMC monolayer increased and cell survival decreased with 0.002% BAC (p < 0.05). Increased permeability without decreasing cell survival occurred with 0.05 μ g/mL MMC. No effect on the permeability or cell survival was observed at 0.1 or 1.0 μ m DEX (p > 0.05).

Conclusions

BAC and MMC induced cellular toxicity and stress at lower concentrations but did not affect survival of cultured HTMCs.

Keywords: Benzalkonium chloride, Carboxyfluorescein, Dexamethasone, Mitomycin C, Trabecular meshwork cells

References

1. Alvarado J, Murphy C, Juster R. Trabecular meshwork cellularity in primary open-angle glaucoma and nonglaucomatous normals. Ophthalmology. 1984; 91:564–79.

2. Rohen JW, Lütjen-Drecoll E, Flügel C. . Ultrastructure of the trabecular meshwork in untreated cases of primary open-angle glaucoma (POAG). Exp Eye Res. 1993; 56:683–92.

3. Hamard P, Blondin C, Debbasch C. . In vitro effects of preserved and unpreserved antiglaucoma drugs on apoptotic marker expression by human trabecular cells. Graefes Arch Clin Exp Ophthalmol. 2003; 241:1037–43.

4. Yee RW. The effect of drop vehicle on the efficacy and side effects of topical glaucoma therapy: a review. Curr Opin Ophthalmol. 2007; 18:134–9.

5. Wilhelmus KR. The Draize eye test. Surv Ophthalmol. 2001; 45:493–515.

6. Doucet O, Lanvin M, Thillou C. . 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. 2006; 20:499–512.

7. Khoh-Reiter S, Jessen BA. Evaluation of the cytotoxic effects of ophthalmic solutions containing benzalkonium chloride on corneal epithelium using an organotypic 3-D model. BMC Ophthalmol. 2009; 9:5.

8. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65:55–63.

9. Freimoser FM, Jakob CA, Aebi M, Tuor U. The MTT [3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities. Appl Environ Microbiol. 1999; 65:3727–9.

10. Grimes PA, Stone RA, Laties AM, Li W. Carboxyfluorescein. A probe of the blood-ocular barriers with lower membrane permeability than fluorescein. Arch Ophthalmol. 1982; 100:635–9.

11. Araie M. Carboxyfluorescein. A dye for evaluating the corneal endothelial barrier function in vivo. Exp Eye Res. 1986; 42:141–50.

12. Araie M. Barrier function of corneal endothelium and the intra-ocular irrigating solutions. Arch Ophthalmol. 1986; 104:435–8.

13. Tsuboi S, Pederson JE. Permeability of the isolated dog retinal pigment epithelium to carboxyfluorescein. Invest Ophthalmol Vis Sci. 1986; 27:1767–70.

14. Blair NP, Rusin MM. Blood-retinal barrier permeability to carbox-yfluorescein and fluorescein in monkeys. Graefes Arch Clin Exp Ophthalmol. 1986; 224:419–22.

15. Grimes PA. Carboxyfluorescein transfer across the blood-retinal barrier evaluated by quantitative fluorescence microscopy: comparison with fluorescein. Exp Eye Res. 1988; 46:769–83.

16. Grimes PA. Carboxyfluorescein distribution in ocular tissues of normal and diabetic rats. Curr Eye Res. 1988; 7:981–8.

17. Kimura M, Araie M, Koyano S. Movement of carboxyfluorescein across retinal pigment epithelium-choroid. Exp Eye Res. 1996; 63:51–6.

18. Lei Y, Stamer WD, Wu J, Sun X. Oxidative stress impact on barrier function of porcine angular aqueous plexus cell monolayers. Invest Ophthalmol Vis Sci. 2013; 54:4827–35.

19. Burke AG, Zhou W, O'Brien ET. . Effect of hydrostatic pressure gradients and Na2EDTA on permeability of human Schlemm's canal cell monolayers. Curr Eye Res. 2004; 28:391–8.

20. Nakagawa S, Usui T, Yokoo S. . Toxicity evaluation of antiglaucoma drugs using stratified human cultivated corneal epithelial sheets. Invest Ophthalmol Vis Sci. 2012; 53:5154–60.

21. Razeghinejad MR, Katz LJ. Steroid-induced iatrogenic glaucoma. Ophthalmic Res. 2012; 47:66–80.

22. Sibayan SA, Latina MA, Sherwood ME. . Apoptosis and morphologic changes in drugtreated trabecular meshwork cells in vitro. Exp Eye Res. 1998; 66:521–9.

Figure 1.

Effects of benzalkonium chloride (BAC) on the survival of trabecular meshwork cells. BAC concentrations more than 0.002% decreased cellular viability significantly (^* p < 0.05). PBS = phosphate buffered saline.

Figure 2.

Effects of mitomycin C (MMC) on the survival of trabecular meshwork cells. 0.5 μ g/mL MMC decreased cellular viability significantly (^* p < 0.05). PBS = phosphate buffered saline.

Figure 3.

Effects of dexamethasone (DEX) or 0.06% ethanol (vehicle) on the survival of trabecular meshwork cells. Both 0.1 or 1.0 μ m DEX showed no significant effects on the cellular viability (p > 0.05). PBS = phosphate buffered saline.

Figure 4.

Effects of benzalkonium chloride (BAC) on the permeability of carboxyfluorescin through the trabecular mesh-work cell monolayer. 0.002% BAC increased permeabilty of carboxyfluorescein significantly (^* p < 0.05). Carboxyfluorescein intensity of outer chamber normalized to the mean value obtained using PBS (permeability 100%). PBS = phosphate buffered saline.

Figure 5.

Effects of mitomycin C (MMC) on the permeability of carboxyfluorescin through the trabecular meshwork cell monolayer. 0.05 μ g/mL MMC increased permeabilty of car-boxyfluorescein significantly (^* p < 0.05). Carboxyfluorescein intensity of outer chamber normalized to the mean value obtained using PBS (permeability 100%). PBS = phosphate buffered saline.

Figure 6.

Effects of dexamethasone (DEX) on the permeability of carboxyfluorescin through the trabecular meshwork cell monolayer. Both 0.1 or 1.0 μ m DEX showed no significant effects on the permeabilty of carboxyfluorescein (p > 0.05). Carboxyfluorescein intensity of outer chamber normalized to the mean value obtained using PBS (permeability 100%). PBS = phosphate buffered saline.

TOOLS

Similar articles