br Conclusion GST expression was detected in the Hemicornea

Conclusion GST expression was detected in the Hemicornea construct and the commonly used animal cornea models at both the protein and functional levels. The results are summarized in Table 1. However, the construct exhibited lower levels of activity of GST, a marker of phase II enzymatic activity, compared with those of the porcine and rabbit corneas. Evaluation of the results obtained in this study is challenging because knowledge about glutathione transferase expression in the human cornea is scarce. This study demonstrated differences in the absolute as well the relative levels of glutathione transferase expression in the layers of porcine and rabbit corneas. The observed inconsistency in the levels of glutathione transferase activity in the layers of these models demonstrates the difficulty of transferring data from one mammalian organ to another. Therefore, assuming that the findings obtained from experimentation using animals apply to humans is questionable. Further studies of the human cornea should be performed to confirm the results obtained in this investigation.
Acknowledgements We are grateful to the German Federal Institute for Risk Assessment (BfR) for funding this study with grant no. FK 3‐1328‐306‐52054369. The authors thank Dr. K. Araki‐Sasaki (Kagoshima, Japan) and Dr. M. Zorn‐Kruppa (UKE Hamburg, Germany) for their generous gifts of the HCE‐T and HCK cell lines. We further gratefully acknowledge Joana Bartels (Institute of Pharmacology, Toxicology and Clinical Pharmacy, Technische Universität Braunschweig, Braunschweig, Germany) for her support in performing the confocal microscope measurements.
The GSTs(EC 2.5.1.18) are a multigene family of isozymes that catalyze the conjugation with glutathione of many toxic, electrophilic compounds , , . Generally, but not always, the conjugates formed are less toxic to the cell. Consequently, GSTs are believed to play important roles in the protection of Paxilline australia from these toxic substrates that include antineoplastic drugs, carcinogens, and mutagens , , . Increased levels of GSTs are often associated with the emergence of resistance to some antineoplastic agents in drug-selected cell lines , , . To obtain more direct evidence in support of the role of GST isozymes in drug resistance, GST expression vectors were stably transfected into MCF7 cells—cells that normally express very low levels of GST. In these studies, increases in cytosolic GSTs generally failed to confer significant resistance to antineoplastic drugs, including drugs known to be substrates of the GST isozymes transfected , , . These results led to the suggestion that other factors necessary to potentiate GST-mediated resistance may be limiting in MCF7 cells . These co-factors of resistance might include glutathione levels or GS-X export. Indeed, conjugates of glutathione and their metabolites, if not efficiently transported out of the cells, may themselves be cytotoxic—either directly or by inhibition of other glutathione-dependent reactions , , , , . Recently, MRP, an energy-dependent, membrane-associated drug efflux pump responsible for one form of P-glycoprotein-independent MDR, has been shown to support the efflux of several conjugates of glutathione , , , , . The finding that MRP is an important GS-X transporter led us to propose that coordinated expression of MRP and GST may confer a level of resistance to some cytotoxic compounds that is greater than that achievable by the expression of either protein alone. In this view, drug detoxification would involve the sequential formation of drug–glutathione conjugates in GST-catalyzed reactions followed by conjugate efflux by MRP-mediated transport. Accordingly, the failure of increased GST isozymes to confer resistance in transfected MCF7 cells to the drugs tested previously may have been due to the extremely low level of MRP expressed in these cells . Indeed, even though the human pi class GST, GSTP1-1, efficiently catalyzes the conjugation of 4-nitroquinoline 1-oxide with glutathione, we recently showed that high level protection from the cytotoxicity of 4-nitroquinoline 1-oxide by GSTP1-1 in MCF7 cells is attained only when MRP is expressed concomitantly with GSTP1-1 , .