Toxicity of natural tear substitutes in a fully defined. culture model of human corneal epithelial cells

PURPOSE. Serum and saliva have recently been advocated as natural rear subs titutes for intractable aqueous-deficient dry eyes, but the effects of thes e fluids on corneal epithelium have not been well characterized. A laborato ry study was performed in a defined test model to compare the toxicity of n atural and pharmaceutical tear substitutes and to identify potentially toxi c factors in natural tear substitutes, such as amylase. hypotonicity, and v ariations 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 v ol/vol with chloramphenicol 0.5%) for varying times and concentrations. Tox icity was examined in four ways. Microvillous density was assessed with sca nning electron microscopy. Cell membrane permeability and intracellular est erase activity were analyzed after staining with fluorescent calcein-AM/eth idium homodimer and cellular adenosine triphosphate (ATP) was quantified us ing a luciferin-luciferase- based assay. RESULTS. The toxicity ranking of the tear substitutes correlated in all ass ays. The ATP assay was the most sensitive, followed by ethidium cell permea bility, and finally the esterase activity. Preserved hypromellose was more toxic than the unpreserved preparation. Among natural tear substitutes, nat ural saliva was most toxic. Isotonic saliva and 50% serum were of similar t oxicity, and 100% serum was least toxic. Natural tear substitutes were-exce pt for natural saliva-less toxic than unpreserved hypromellose. Hypotonicit y but not amylase, was the major toxic effect associated with saliva. The d ilution of serum with chloramphenicol induced toxicity. CONCLUSIONS. This is the first toxicity study using human primary corneal e pithelial cells cultured under fully defined conditions as an in vitro mode l. 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.