Mutation Research 772 (2015) 1–9 Contents lists available at ScienceDirect Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis j ourna l h om epage: www.elsevier.com/l ocate/molmut Comm unit y ad dress: www.elsevier.com/locate/mutres Genomic and post-genomic effects of anti-glaucoma drugs preservatives in trabecular meshwork Alberto Izzotti a,b,∗ , Sebastiano La Maestra b , Rosanna Tindara Micale b , Maria Grazia Longobardi b , Sergio Claudio Saccà c a Mutagenesis Unit, IRCCS AOU San Martino – IST, Genova, Italy b Department of Health Sciences, University of Genoa, Via A. Pastore 1, 16132 Genoa, Italy c Ophthalmology Unit, IRCCS AOU San Martino-IST, Genova, Italy a r t i c l e i n f o Article history: Received 4 July 2014 Received in revised form 19 November 2014 Accepted 21 November 2014 Available online 18 December 2014 Keywords: Drug preservatives Glaucoma Trabecular meshwork Oxidative damage Microarray Gene expression a b s t r a c t Oxidative stress plays an important role in glaucoma. Some preservatives of anti-glaucoma drugs, com- monly used in glaucoma therapy, can prevent or induce oxidative stress in the trabecular meshwork. The aim of this study is to evaluate cellular and molecular damage induced in trabecular meshwork by preservatives contained in anti-glaucoma drugs. Cell viability (MTT test), DNA fragmentation (Comet test), oxidative DNA damage (8-oxo-dG), and gene expression (cDNA microarray) have been evaluated in trabecular meshwork specimens and in human trabecular meshwork cells treated with benzalkonium chloride, polyQuad, purite, and sofzia-like mixture. Moreover, antimicrobial effectiveness and safety of preservative contents in drugs was tested. In ex vivo experiments, benzalkonium chloride and polyQuad induced high level of DNA damage in trabecular meshwork specimens, while the effect of purite and sofzia were more attenuated. The level of DNA fragmentation induced by benzalkonium chloride was 2.4-fold higher in subjects older than 50 years than in younger subjects. Benzalkonium chloride, and polyQuad significantly increased oxidative DNA damage as compared to sham-treated specimens. Gene expression was altered by benzalkonium chloride, polyQuad, and purite but not by sofzia. In in vitro experiments, benzalkonium chloride and polyQuad dramatically decreased trabecular meshwork cell viability, increased DNA fragmentation, and altered gene expression. A lesser effect was also exerted by purite and sofzia. Genes targeted by these alterations included Fas and effector caspase-3. The efficacy of the preservatives in inhibiting bacterial growth increased the adverse effects in trabecular meshwork in terms of DNA damage and alteration of gene expression. Presented data indicates the delicate balance between efficacy and safety of drug preservatives as not yet optimized. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Our previous study demonstrated that oxidative damage occurs at a high level in the trabecular meshwork (TM) of glaucomatous patients [1] and is related to the extent of intra-ocular pressure alteration and visual field damage [2]. Oxidative damage target- ing TM in glaucoma is due to the high sensitivity of this tissue to oxidative stress as compared to the other components of the ocular Abbreviations: TM, trabecular meshwork; HTM, human trabecular meshwork; BKA, Benzalkonium chloride; PLYQ, polyquad; SFZ, sofzia like mixture; PRT, purite; 8-oxo-dG, 8-hydroxy-2 ′ -deoxyguanosine. ∗ Corresponding author at: Mutagenesis Unit, IRCCS AOU San Martino – IST, Genova, Italy. Tel.: +39 0103538522; fax: +39 0103538504. E-mail address: izzotti@unige.it (A. Izzotti). anterior chamber [3]. Evidence has been provided that aqueous humor protein composition undergoes dramatic alterations during glaucoma course mainly oriented toward decrease of antioxidant defenses accompanied by increasing levels of proteins resulting from the damage of TM cells and their intracellular components [4]. Mitochondria damage has a pivotal role as a source of endoge- nous oxidative stress in the TM of glaucomatous patients [5,6]. These findings underline the importance of oxidative damage as occurring in the TM for glaucoma pathogenesis. Accordingly, it is of interest to evaluate whether or not anti-glaucoma drugs can affect the oxidative damage occurring in the ocular anterior chamber. It has been reported that treatment of mammalian cells with common drug preservatives may result in the occurrence of severe oxidative DNA damage and toxicity [7]. A drug com- monly used in glaucoma therapy, i.e. timolol, displays antioxidant effects protecting endothelial cells from oxidative stress [8]. This http://dx.doi.org/10.1016/j.mrfmmm.2014.11.006 0027-5107/© 2014 Elsevier B.V. All rights reserved.