DETOXICATION OF XENOBIOTICS IN ERYTHROCYTES

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In human tissues such as liver and kidney, the mercapturic acid pathway serves as one of the important means of detoxifying the xenobiotics or foreign chemicals. However, human erythrocytes do not have complete mercapturic acid pathway. The erythrocytes contain high levels of the first enzyme of the mercapturic acid pathway, glutathione S-transferase, which conjugates glutathione (GSH) to a large number of the hydrophobic xenobiotics carrying and electrophylic center. We have recently demonstrated that the conjugate of 1-chlore-2,4-dinitrobenzene (CDNB) with GSH is formed in erythrocytes. This conjugate is not metabolized further within these cells and is actively transported out of the erythrocytes. We propose to study the mechanism of transport of the conjugates of GSH and xenobiotics from human erythrocytes. Glutathione S-transferase will be purified to homogeneity from human erythrocytes. The purified enzyme will be studied for its kinetic properties towards various xenobiotics and will also be used for the synthesis of conjugates of GSH and xenobiotics which will be used in transport studies. The kinetics of the transport of the conjugates will be studied in normal and glucose-6-phosphate dehydrogenase deficient erythrocytes, resealed ghosts and inside out vesicles formed from erythrocytes. In order to better understand the mechanism of the transport of the conjugates, the transporter protein will be purified and the transport will be studied in the reconstituted proteoliposomes. Since oxidized glutathione (GSSG) is also actively transported from the erythroctyes, we will study whether or not both GSSG and GSH-xenobiotic conjugates are transported via the same transporter protein. The proposed studies will be of great significance in understanding the mechanisms by which erythrocytes detoxify xenobiotics and environmental pollutants.