GSH MEDIATED MECHANISMS IN NITROSOUREA TOXIC EFFECTS

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One of the consequences of the therapeutic use of BCNU is the inactivation of GSSG reductase. There is mounting evidence that the inactivation of this key antioxidant enzyme, leading to the cellular accumulation of toxic levels of GSSG, is a major factor in pulmonary and cerebral toxicities of BCNU. This project explores the molecular consequences of a BCNU-GSH derived inactivator of GSSG reductase. Preliminary studies revealed that an active site-directed inactivator of GSSG reductase, designated GS-I, was formed by the reaction of a BCNU metabolite with GSH. GS-I will be chemically synthesized and its structure proven by FAB mass spectrometry and 500 MHz NMR analyses. The kinetics and stoichiometry of the inactivation of purified human erythrocyte GSSG reductase by GS-I will be studied. Intact erythrocytes will be used to determine if exogenous GS-I inactivates erythrocytic GSSG reductase. It will also be determined if GS-I is synthesized and secreted by erythrocytes. Studies are designed to establish whether GSH transferase isoenzymes from human liver and erythrocytes catalyze the synthesis of GS-I from BCNU or limit the formation of GS-I by catalyzing the conversion of BCNU to other products, and whether such products are inactivators of GSSG reductase. The proposed structure for GS-I strongly suggests that it may serve as an active site-directed inactivator of other GSH- and GSSG-linked enzymes, which may then be additional specific molecular targets for BCNU toxic effects. The intrinsic susceptibilities to GS-I of purified gamma- glutamyltranspeptidase, GSH transferase isoenzymes, and glyoxalases I and II will be examined. Whether GS-I is a substrate for hydrolysis by glyoxalase II and/or by gamma-glutamyltrans- peptidase will also be investigated. This project is expected to (a) reconcile the in vitro and in vivo observations of others concerning the basis of BCNU-induced GSSG reductase deficiency, (b) increase understanding of nitrosourea activation and inactivation via GSH-dependent pathways, (c) reveal additional specific molecular targets of BCNU toxicities, and (d) provide information that will aid in predicting toxic effects of new nitrosoureas while these drugs are still at the design stage.