Effect of hydration on opacity in the bovine corneal opacity and permeability (BCOP) assay

The bovine corneal opacity and permeability (BCOP) assay has been proposed an an in vitro method for predicting the irritancy or toxicity of chemical substances. A loss of corneal transparency, which reduces visual acuity, ca n be the result of an increase in corneal hydration or direct damage to cor neal tissues. The BCOP assay as currently practiced does not differentiate between these mechanisms. The purpose of this study was to determine the ef fect of increased hydration on transparency of the bovine cornea and to mea sure the hydration levels of corneas treated with compounds and substances known, from previous BCOP studies, to cause corneal opacity. Corneas were t reated according to the BCOP protocol and corneal opacity was determined by measurement of light absorbance at 570 nm (A(570)) Corneal hydration was d etermined by measurement of wet and dry weights. Hydration of the intact co rnea was 3.86 mg H2O/mg dry weight and A(570) = 0.048. After removal of the epithelium and incubation in MEM, corneal hydration increases to 6.27 mg H 2O/mg and A(570) = 0.11. Hydration of the maximally hydrated cornea, follow ing removal of the epithelium and endothelium and incubation in deionized w ater, is 16.33 mg H2O/mg and A(570) = 0.67. The hydration of corneas treate d with compounds that are ranked as moderate to severe irritants based on t he BCOP assay is not correlated with hydration levels of these corneas, sin ce the A(570) Of these corneas was usually higher than predicted from hydra tion levels. For example, after exposure to isopropanol hydration = 4.7, A( 570) = 0.59; acetone: hydration = 5.69, A(570) = 1.38; 30% trichloroacetic acid: hydration = 4.28, A(570) = 1.43; 1% NaOH: hydration = 8.22, A(570) = 1.7. In contrast, after exposure to 30% sodium lauryl sulfate (SLS), which only removes epithelium, hydration = 5.38, A(570) = 0.095. This suggests th at opacity in excess of that caused by increased hydration is caused by dam age to corneal tissue, including epithelium and stroma. This was confirmed by light and transmission electron microscopy. Determining the cause of an increase in corneal opacity in the BCOP assay is important since tissue dam age may be irreversible, while the cornea can recover from moderate increas es in hydration. On the other hand, since increases in hydration alone caus e relatively low A(570) readings, a potential irritant that causes a visual ly significant increase in cornea hydration might be ranked as a mild irrit ant. It is therefore proposed that measurement of corneal hydration be adde d to the BCOP protocol.