TOXICI TY OF ANTIBIOTICS AND ANTIFUNGALS ON CULTURE D HUMAN CORNEAL CELLS: EFFEC T OF MIXING, EXPOSURE AND CONCENTRATION M. BERRY, A. GURUNG and D. L. EASTY Bristol SUMMARY effective treatment of bacterial and fungal conjuncti­ Toxic effects of topical drugs may be masked by vitis or keratitis. The external eye surface may be manifestations of the disease they cure. The toxicity exposed to broad spectrum drugs or drug combina­ of drug mixtures has not been thoroughly studied. We tions to control multiple or unidentified infective therefore investigated cytopathic effects on primary agents. Appearance of local toxic effects of the cultures of human corneal cells of six topical anti­ medication, often difficult to distinguish from the microbials singly and in combinations of any two, to disease process itselC may prompt a diminution of determine the combined toxicity ranking and the drug concentration or frequency or a change of interaction between duration of exposure and concen­ medication. tration. Preconfluent cultures were exposed to fixed Toxicity of drug combinations, resulting from a dilutions of single drugs, or to equal-dilution mixtures summation or potentiation of individual adverse effects, of two drugs, for 7 and 14 days. Diminishing has been insufficiently studied. It is this relationship concentrations of single drugs were applied sequentially between the toxicity of a drug mixture and that of its to cultures for 14 days. The number of metabolically components that is the subject of this study. competent cells was assessed by measuring hexosami­ The number of all possible combinations of drugs nidase and total protein. Toxic effects depended on is too large to be exhaustively tested. We tested, substance, concentration and exposure. The scale of singly and in combinations of any two, three toxicity determined for single drugs after 7 days of antibiotics and three antifungals most frequently exposure was: gentamicin > econazole � methicillin � prescribed in the Bristol Eye Hospital: chloramphe­ c1otrimazole miconazole chloramphenicol. After � � nicol, gentamicin, methicillin, clotrimazole, econa­ 14 days this order changed: in particular chloramphe­ zole and miconazole. nicol showed a highly increased toxicity. The order of An in vitro system was chosen to isolate drug diminishing effects was: gentamicin> chloramphenicol toxicity from cytopathic effects of a microbial � methicillin > miconazole > econazole > c1otrima­ zole. A clear reduction in cytopathic effects was infection. Human corneal epithelial cell cultures observed when drug concentration was decreased were exposed to antimicrobial drugs for a period progressively only in cultures treated with gentamicin commensurate with clinical use. They were exposed or methicillin. All drug combinations were more toxic to fixed and to sequentially decreasing concentra­ than their components at equal dilution. Combinations tions of test substance, the latter mimicking a containing chloramphenicol ranked most toxic overall, tapering off regime. those containing econazole least. A tapering off Cytopathic effects depended on the drug(s), combination regime did not improve cell survival. concentration and duration of exposure. Rankings These in vitro toxicity data complement clinical studies of single drugs and combinations were not corre­ and suggest ways in which topical drugs can be chosen lated. Exposure to diminishing drug concentrations to minimise toxic effects to corneal surface. did not always improve the outcome. Topical antimicrobial drugs are a widespread and MATERIALS AND METHODS Selection of Topical Drugs Correspondence to: Dr M. Berry, University of Bristol, Department of Ophthalmology, Bristol Ey e Hospital, Lower Twenty-four questionnaires were sent to consultants, Maudlin Street, Bristol BSI 2LX, UK. registrars and senior house officers in the Bristol Eye Eye (1995) 9, 110-115 © 1995 Royal College of Ophthalmologists ANTI MICROBIALS IN VITRO 111 Hospital. They were asked to list in order of test substance for 3 days. On day 3 the concentration preference drugs (and combinations) for treating was halved (Cf2) for all but 6 wells, which were suspected bacterial and fungal conjunctivitis or exposed to the initial concentration for the remain­ keratitis. der of the experiment, as a fixed concentration comparator. On day 6 the concentration was halved Tissue Culture again (Cf4) for all but another block of 6 wells which continued to be exposed at half the initial concentra­ Epithelial cell cultures were established from human tion (Cf2). This process was repeated every 3 days, corneas unsuitable for transplantation from the resulting in different concentration step length UKTSSA Eye Bank in Bristol. Epithelium and a X regimes. The number of metabolically competent thin layer of underlying stroma was separated from cells was determined in all the cultures on day 21. the main bulk of the stroma by blunt dissection. Analysis of variance (ANOVA), post hoc tests and Explants 2-4 mm2 were plated on tissue culture non-parametric analysis of variance by ranks (Krus­ flasksin RPMI 1640 (Gibco) supplemented with 10% kal-Wallis test) were performed with Stat-View 4.02 fetal bovine serum (Gibco), streptomycin, penicillin, (Abacus Concepts, Berkeley, CA). amphotericin mixture (Sigma) and RPMI nonessen­ tial amino-acids (Sigma). When epithelial growths had been established explants were removed to RESULTS minimise fibroblast content of the cultures. After Thirteen of 24 questionnaires were returned. The confluence cultures were passaged and the cells used unanimous first choice of drug for the treatment of in this study. All experiments were carried out in six suspected bacterial conjunctivitis was chlorampheni­ replicates in tissue culture medium containing 5% col 0.5%. No drug combinations were envisaged for fetal bovine serum. this condition. For suspected bacterial keratitis gentamicin (1.5%) or a combination of gentamicin Experimental Design (1.5% or 0.3%) and methicillin 2% were equal first preferences. Suspected fungal keratitis would most Effects on Cell Proliferation. A total of 5 103 cells X often be treated with either miconazole 1%, or a per well were seeded in 96-well tissue culture plates mixture of two antifungals (e.g. econazole plus (Costar), allowed to adhere overnight at 35 °C, and miconazole), or a mixture of an antibiotic and an exposed to a fixedconcentration of test substance for antifungal (e.g. econazole plus chloramphenicol, or 7 or 14 days. Water-soluble topical solutions were clotrimazole plus gentamicin forte). diluted in tissue culture medium, lipid-soluble ones in In accordance to these preferences we chose to arachis oil (Hill Cross Pharmaceuticals). The latter study the effects of chloramphenicol 0.5% (Scher­ were always applied to the cell layer before tissue ring-Plough), gentamicin forte 1.5% (MoorfieldsEye culture medium to ensure penetration. Cells treated Hospital) and methicillin 2% (Bristol Eye Hospital). with arachis oil and tissue culture medium served as The antifungals chosen were econazole 1% (Moor­ controls for each experiment. Medium and test fields Eye Hospital), miconazole 1 % (Moorfields substance were renewed twice a week. The number Eye Hospital) and clotrimazole 1 % (Moorfields Eye of cells was evaluated by measuring hexosaminidase Hospital). activitr and total protein content (BCA Protein The number of metabolically competent cells as Assay, Pierce). The results are presented as optical evaluated by the hexosaminidase assay paralleled densities, with standard curve number of cells closely the results obtained by measuring total corresponding to that optical density on the right­ protein. Hexosaminidase results had, however, a hand vertical axis. wider distribution, i.e. a larger difference in optical Effects on Cell Proliferation and Migration. Cells density between the most and the least populated were plated overnight either in a dense plaque (5 x wells (Fig. 1). We chose the latter to represent the 103 cells per well) in the centre of a 1.5 cm diameter effects of treatment on cell numbers. well (Costar), or in an annulus (15 X 103 cells per well) around its perimeter, and exposed to test Effect on Cell Proliferation solutions for 14 days. The number of cells was After exposure to antimicrobials the number of cells evaluated by measuring hexosaminidase activity and depended on test substance, concentration and total protein, and well cover visualised by staining duration of exposure (Kruskal-Wallis analysis of with haematoxylin. variance by ranks, p<O.OOI; Figs. 2 and 3). Effects of Decreasing Concentration. Step decreases Fourteen days of exposure to antibiotics brought a in the concentration of the test substance (single further decrease in population size compared with drugs or combinations) were compared with expo­ the shorter exposure (Fig. 3). This effect was not seen sure to fixed concentrations. Cells plated in 96-well wi th antifungals. plates were exposed to a highest concentration (C) of After 7 days of exposure topical solutions ranked 112 M. BERRY ET AL. 1.4 1.2 -r-��-�--'--��-�--'-T 1.2 1 ...... Chloramphenicol -a- Clotrtmazole i·8 -- Econazole -8].6 @] Hexosaminidase ....... Gentamicin Forte 1m Total protein ___ MethJcUlin 4 ___ Mlconazole 8-..2 � .2 0 -.2 ISO BO 40 20 10 5 4 2 2 4 5102040 BO dilution of topical solution dUutlon factor of topical solution Fig. Dependence of effect on substance and concentra­ Fig. Comparison between hexosaminidase and total 2. 1. tion. Analysis of variance (optical