PURPOSE: Serum and saliva have recently been advocated as natural tear substitutes for intractable aqueous-deficient dry eyes, but the effects of these fluids on corneal epithelium have not been well characterized. A laboratory study was performed in a defined test model to compare the toxicity of natural and pharmaceutical tear substitutes and to identify potentially toxic factors in natural tear substitutes, such as amylase, hypotonicity, and variations in preparation. METHODS: Primary human corneal epithelial cells were cultured with defined keratinocyte serum-free medium. The cells were incubated with hypromellose (hydroxypropylmethylcellulose 0.3%) with and without benzalkonium chloride 0.01%, saliva with differing osmolalities, 100% serum, and 50% serum (1:1 vol/vol with chloramphenicol 0.5%) for varying times and concentrations. Toxicity was examined in four ways. Microvillous density was assessed with scanning electron microscopy. Cell membrane permeability and intracellular esterase activity were analyzed after staining with fluorescent calcein-AM/ethidium homodimer and cellular adenosine triphosphate (ATP) was quantified using a luciferin-luciferase-based assay. RESULTS: The toxicity ranking of the tear substitutes correlated in all assays. The ATP assay was the most sensitive, followed by ethidium cell permeability, and finally the esterase activity. Preserved hypromellose was more toxic than the unpreserved preparation. Among natural tear substitutes, natural saliva was most toxic. Isotonic saliva and 50% serum were of similar toxicity, and 100% serum was least toxic. Natural tear substitutes were-except for natural saliva-less toxic than unpreserved hypromellose. Hypotonicity, but not amylase, was the major toxic effect associated with saliva. The dilution of serum with chloramphenicol induced toxicity. CONCLUSIONS: This is the first toxicity study using human primary corneal epithelial cells cultured under fully defined conditions as an in vitro model. Cellular ATP is a sensitive parameter for quantifying toxicity. Isotonic saliva and serum offer greater therapeutic potential for severely aqueous-deficient dry eyes than do pharmaceutical tear substitutes.
PURPOSE: Serum and saliva have recently been advocated as natural tear substitutes for intractable aqueous-deficient dry eyes, but the effects of these fluids on corneal epithelium have not been well characterized. A laboratory study was performed in a defined test model to compare the toxicity of natural and pharmaceutical tear substitutes and to identify potentially toxic factors in natural tear substitutes, such as amylase, hypotonicity, and variations in preparation. METHODS: Primary human corneal epithelial cells were cultured with defined keratinocyte serum-free medium. The cells were incubated with hypromellose (hydroxypropylmethylcellulose 0.3%) with and without benzalkonium chloride 0.01%, saliva with differing osmolalities, 100% serum, and 50% serum (1:1 vol/vol with chloramphenicol 0.5%) for varying times and concentrations. Toxicity was examined in four ways. Microvillous density was assessed with scanning electron microscopy. Cell membrane permeability and intracellular esterase activity were analyzed after staining with fluorescent calcein-AM/ethidium homodimer and cellular adenosine triphosphate (ATP) was quantified using a luciferin-luciferase-based assay. RESULTS: The toxicity ranking of the tear substitutes correlated in all assays. The ATP assay was the most sensitive, followed by ethidium cell permeability, and finally the esterase activity. Preserved hypromellose was more toxic than the unpreserved preparation. Among natural tear substitutes, natural saliva was most toxic. Isotonic saliva and 50% serum were of similar toxicity, and 100% serum was least toxic. Natural tear substitutes were-except for natural saliva-less toxic than unpreserved hypromellose. Hypotonicity, but not amylase, was the major toxic effect associated with saliva. The dilution of serum with chloramphenicol induced toxicity. CONCLUSIONS: This is the first toxicity study using human primary corneal epithelial cells cultured under fully defined conditions as an in vitro model. Cellular ATP is a sensitive parameter for quantifying toxicity. Isotonic saliva and serum offer greater therapeutic potential for severely aqueous-deficient dry eyes than do pharmaceutical tear substitutes.
Authors: L Liu; D Hartwig; S Harloff; P Herminghaus; T Wedel; G Geerling Journal: Graefes Arch Clin Exp Ophthalmol Date: 2005-03-09 Impact factor: 3.117