Interestingly the rabbit cornea exhibited a

Interestingly, the rabbit cornea exhibited a more than two-fold higher level of total GST activity compared with that of the porcine cornea, whereas their levels of GSTO1‐1 and GSTP1‐1 activity were similar (Fig. 4). Because members of the GST Alpha, Mu, Pi and Omega FPH1 primarily utilize CDNB as a substrate, the levels of Alpha and Mu GST expression may be different. However, the GST expression pattern might depend on the thickness of the cornea. The rabbit cornea is thinner than the porcine cornea, which are approximately 382-μm thick (Li et al., 1997) and 666-μm thick (Faber et al., 2008), respectively. In addition, the epithelial layer of both the rabbit and porcine corneas comprise approximately 10% of the corneal thickness (Jay et al., 2008, Li et al., 1997). Therefore, it might be presumed that the thickness of these corneal layers would correlate with the relative levels of GST metabolic activity. However, this is not the case. The levels of GST activity in corneal tissues are not necessarily constant. The expression of members of the Alpha, Mu, Pi and Theta GST classes was found to be inducible in the rat liver (Hayes and McLellan, 1999). Oxidative stress generally appears to increase GST activity (Hayes and McLellan, 1999). Therefore, a xenobiotic such as phenobarbital, which is metabolized by cytochrome P450 enzymes, can induce GST activity by generating reactive oxygen species (Aniya et al., 1993). Ultraviolet radiation, such as that used for excimer laser keratectomy or even uv radiation of the daylight, can raise GST activity levels (Bilgihan et al., 1998, Svobodová et al., 2011). In addition, corneal disorders, such as keratoconus or herpes keratitis can increase GST activity relative to that of healthy corneas (Gondhowiardjo and van Haeringen, 1993). The exposure to daylight and the possibility of corneal disorders may help to explain why animal models exhibit higher GST activity levels, compared to the Hemicornea construct, which was cultivated under aseptic and mostly dark conditions. However, the level of GSTP1‐1 activity observed in the human cornea is more similar to that of the Hemicornea construct than to those of the animal models. This finding may contradict that expected based on the considerations mentioned above and it must be noted that it is based on roughly calculated values. Therefore, further investigation of GSTP1‐1 activity is necessary. In general, little is known about the effect of corneal glutathione transferases on ophthalmic drugs. Commonly prescribed ophthalmic drugs, such as timolol and latanoprost, are primarily metabolized by phase I enzymes (Sjöquist and Stjernschantz, 2002, Volotinen et al., 2011). However, the efficacy of these drugs is affected by the preservatives used in eye drops. Thimerosal, which was formerly widely used as a preservative in ophthalmic solutions, is known to inhibit GST activity (Dierickx, 1985). Dierickx measured the total GST activity using the method described by Habig et al., employing 1mM CDNB and 1mM glutathione as substrates (Habig et al., 1974). At a concentration of 100 mM, which was 10% of the substrate concentration, thimerosal decreased the GST activity level of a rat liver sample to less than 40% of the control level. Nonetheless, at a concentration of 0.001%, which is commonly utilized in eye drops, thimerosal does not reduce the level of glutathione transferase activity. However, compared with that of the liver, the level of metabolic activity in the cornea is low. Watkins et al. examined the phase II enzymatic activity in the rabbit cornea relative to that of the liver (Watkins et al., 1991). The glutathione transferase activity per mg protein of rabbit liver was found to be four-fold higher than that of the rabbit cornea. The findings of Watkins et al. can be related to the total weight of the liver and cornea [approximately 56 g and 0.07 g, respectively (Lathe and Ricketts, 1964, Watkins et al., 1991)], which indicated that the rabbit liver has a 3×105-fold higher level of activity than that of the rabbit cornea. The direct effect of corneal GST activity on ophthalmic substances has not been described. It is likely that GSTs are important in detoxifying phase I metabolites.