A major goal of this research proposal will be to evaluate the status of glutathione (GSH) and related low molecular weight thiols (LMWT) and their interactions with known embryotoxins/teratogens of environmental and therapeutic importance. The developing rat, during the teratogen and embryotoxin-sensitive stage of early organogenesis (gestational days 9-11), will be studied using the whole embryo culture system in order to allow for experimental accessibility to the intact conceptus and to eliminate confounding maternal hormonal and physiological influences. This evaluation will involve an analysis of routes of GSH (or precursor) entry into the conceptus, consequences of decreased precursor availability (i.e. nutritional factors), determination of alternative GSH synthetic capacities in the visceral yolk sac (VYS) and embryo proper, and overall GSH turnover under normal and stress conditions. Alterations in the different chemical forms of GSH and related LMWT will be evaluated using HPLC techniques, with special attention given to the quantitation and distribution of GSH and cysteine and their protein-and mixed-disulfide forms. Chemical modulation of GSH levels (BSO, menadione, diamide, OTC, etc.) will be aimed at producing different conditions of depletion and oxidative state and will be compared to changes which occur following the addition of the model teratogens/embryotoxins (2-nitrosofluorene, 7-OH-acetylaminofluorene, acetaminophen, and valproic acid). The chosen compounds represent known in vitro dysmorphogens which differ with respect to metabolism, potential interactions with GSH (adduct formation will be measured) and the overlapping spectrum of abnormalities each produce. Specific changes in intracellular GSH and LMWT status in the embryo and VYS will be correlated with the morphological abnormalities produced by each compound in vitro to determine whether a particular defect may be related to a common qualitative or quantitative shift in thiol status. Decreased conceptual detoxication capacity due to changes in intracellular GSH/LMWT content or redox state may promote increased embryotoxicity/dysmorphogenesis due to unchecked, production of reactive intermediates and their direct interaction with cellular targets. The final goal of this proposal, however, will be to evaluate the possibility that changes in intracellular GSH/LMWT status induced through environmental (nutritional or physiological) factors, chemical modulation or through detoxication of model, embryotoxins/teratogens are independently sufficient to alter any of several critical metabolic or physiologic processes believed to be regulated by GSH and related LMWT. Effects on glycolysis, NADPH/NADP+ status, pentose phosphate shunt activity, lysosomal proteolysis (histotrophic nutrition) and utilization of GSH-derived cysteine in cysteamine, taurine and CoA biosynthesis will be evaluated. Successful completion of this project should provide valuable information concerning the interaction of environmental and therapeutic agents with embryonic GSH- based detoxication systems and suggest possible points of regulatory impact in altering reproductive outcome.
