Abstract
Purpose
To investigate the toxic effects of benzalkonium chloride (BAC), a preservative commonly used in ophthalmic preparations, on DNA single- and double-strand breaks in immortalized human corneal epithelial cells (HCEs).
Methods
HCEs were treated with BAC in concentrations ranging from 0.00005% to 0.001% for 30 min. Cells were examined immediately after BAC exposure and after 24-h recovery. Alkaline comet assay was used to detect DNA single-strand breaks (SSBs). Immunofluorescence microscope detection of the phosphorylated form of histone variant H2AX (γH2AX) foci indicated DNA double-strand breaks (DSBs). Cell viability was measured by the MTT test.
Results
A significant increase of SSBs, detected by alkaline comet assay, was observed in a dose-dependent manner with BAC exposure in HCEs at concentrations of 0.00005% and higher. Such BAC treatment also exhibited a dose-dependent increase in DSBs, evaluated by number of γH2AX foci. In addition, a significant change in the relative cell survival rate of HCEs was observed after exposure to 0.001% BAC for 30 min. Although the toxic effects of BAC could be partly repaired after 24 h of cell recovery, SSBs and DSBs in HCEs were still present after BAC removal.
Conclusions
The results demonstrated that exposure to BAC in HCEs, even at low concentrations, could induce DNA strand breaks, which were present after BAC removal. Cell survival analysis indicated that BAC-induced DNA damage was correlated with the cytotoxic effects.
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References
Podder SK, Moy KC, Lee VH (1992) Improving the safety of topically applied timolol in the pigmented rabbit through manipulation of formulation composition. Exp Eye Res 54:747–757
Smolen VF, Clevenger JM, Williams EJ, Bergdolt MW (1973) Biophasic availability of ophthalmic carbachol. I. Mechanisms of cationic polymer- and surfactant-promoted miotic activity. J Pharm Sci 62:958–961
Majumdar S, Hippalgaonkar K, Repka MA (2008) Effect of chitosan, benzalkonium chloride and ethylenediaminetetraacetic acid on permeation of acyclovir across isolated rabbit cornea. Int J Pharm 348:175–178
Jaenen N, Baudouin C, Pouliquen P, Manni G, Figueiredo A, Zeyen T (2007) Ocular symptoms and signs with preserved and preservative-free glaucoma medications. Eur J Ophthalmol 17:341–349
Broadway DC, Grierson I, O'Brien C, Hitchings RA (1994) Adverse effects of topical antiglaucoma medication. I. The conjunctival cell profile. Arch Ophthalmol 112:1437–1445
Broadway DC, Grierson I, O'Brien C, Hitchings RA (1994) Adverse effects of topical antiglaucoma medication. II. The outcome of filtration surgery. Arch Ophthalmol 112:1446–1454
Liang H, Baudouin C, Pauly A, Brignole-Baudouin F (2008) Conjunctival and corneal reactions in rabbits following short- and repeated exposure to preservative-free tafluprost, commercially available latanoprost and 0.02% benzalkonium chloride. Br J Ophthalmol 92:1275–1282
Debbasch C, Brignole F, Pisella PJ, Warnet JM, Rat P, Baudouin C (2001) Quaternary ammoniums and other preservatives' contribution in oxidative stress and apoptosis on Chang conjunctival cells. Invest Ophthalmol Vis Sci 42:642–652
Chang SW, Chi RF, Wu CC, Su MJ (2000) Benzalkonium chloride and gentamicin cause a leak in corneal epithelial cell membrane. Exp Eye Res 71:3–10
Cha SH, Lee JS, Oum BS, Kim CD (2004) Corneal epithelial cellular dysfunction from benzalkonium chloride (BAC) in vitro. Clin Experiment Ophthalmol 32:180–184
Geerling G, Daniels JT, Dart JK, Cree IA, Khaw PT (2001) Toxicity of natural tear substitutes in a fully defined culture model of human corneal epithelial cells. Invest Ophthalmol Vis Sci 42:948–956
Guo Y, Satpathy M, Wilson G, Srinivas SP (2007) Benzalkonium chloride induces dephosphorylation of Myosin light chain in cultured corneal epithelial cells. Invest Ophthalmol Vis Sci 48:2001–2008
De Saint JM, Brignole F, Bringuier AF, Bauchet A, Feldmann G, Baudouin C (1999) Effects of benzalkonium chloride on growth and survival of Chang conjunctival cells. Invest Ophthalmol Vis Sci 40:619–630
Morgan SE, Kastan MB (1997) p53 and ATM: cell cycle, cell death, and cancer. Adv Cancer Res 71:1–25
Moll UM, Slade N (2004) p63 and p73: roles in development and tumor formation. Mol Cancer Res 2:371–386
Araki-Sasaki K, Ohashi Y, Sasabe T, Hayashi K, Watanabe H, Tano Y, Handa H (1995) An SV40-immortalized human corneal epithelial cell line and its characterization. Invest Ophthalmol Vis Sci 36:614–621
Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191
Kumaravel TS, Jha AN (2006) Reliable Comet assay measurements for detecting DNA damage induced by ionising radiation and chemicals. Mutat Res 605:7–16
Pilch DR, Sedelnikova OA, Redon C, Celeste A, Nussenzweig A, Bonner WM (2003) Characteristics of gamma-H2AX foci at DNA double-strand breaks sites. Biochem Cell Biol 81:123–129
Baudouin C, Labbe A, Liang H, Pauly A, Brignole-Baudouin F (2010) Preservatives in eyedrops: the good, the bad and the ugly. Prog Retin Eye Res 29: 312–334
Olive PL, Banath JP (2006) The comet assay: a method to measure DNA damage in individual cells. Nat Protoc 1:23–29
Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM (1998) DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 273:5858–5868
Rothkamm K, Lobrich M (2003) Evidence for a lack of DNA double-strand break repair in human cells exposed to very low X-ray doses. Proc Natl Acad Sci USA 100:5057–5062
Burgalassi S, Chetoni P, Monti D, Saettone MF (2001) Cytotoxicity of potential ocular permeation enhancers evaluated on rabbit and human corneal epithelial cell lines. Toxicol Lett 122:1–8
Huhtala A, Mannerstrom M, Alajuuma P, Nurmi S, Toimela T, Tahti H, Salminen L, Uusitalo H (2002) Comparison of an immortalized human corneal epithelial cell line and rabbit corneal epithelial cell culture in cytotoxicity testing. J Ocul Pharmacol Ther 18:163–175
Sancar A (1994) Mechanisms of DNA excision repair. Science 266:1954–1956
Paull TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M, Bonner WM (2000) A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol 10:886–895
Lowndes NF, Toh GW (2005) DNA repair: the importance of phosphorylating histone H2AX. Curr Biol 15:R99–R102
Ward IM, Minn K, Jorda KG, Chen J (2003) Accumulation of checkpoint protein 53BP1 at DNA breaks involves its binding to phosphorylated histone H2AX. J Biol Chem 278:19579–19582
Li Z, Yang J, Huang H (2006) Oxidative stress induces H2AX phosphorylation in human spermatozoa. FEBS Lett 580:6161–6168
Fernandez-Capetillo O, Lee A, Nussenzweig M, Nussenzweig A (2004) H2AX: the histone guardian of the genome. DNA Repair (Amst) 3:959–967
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (NOS. 81070756); the Natural Science Foundation of Zhejiang Province of China (NOS. Y208396); and the International Science and Technology Cooperation Project of Zhejiang Province of China (NOS. 2008C14099).
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No authors have any type of financial interest related to this manuscript.
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The authors have full control of all primary data, and all the authors agree to allow Graefe's Archive for Clinical and Experimental Ophthalmology to review our data upon request.
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Ye, J., Wu, H., Zhang, H. et al. Role of benzalkonium chloride in DNA strand breaks in human corneal epithelial cells. Graefes Arch Clin Exp Ophthalmol 249, 1681–1687 (2011). https://doi.org/10.1007/s00417-011-1755-0
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DOI: https://doi.org/10.1007/s00417-011-1755-0