The role of intracellular thiol oxidation as a possible mechanism for chemically-induced embryotoxicity: Effects of GSH oxidation by diamide.

Abstract

Reduced glutathione (GSH) is known to be involved in a variety of cellular functions. Chemically-induced changes in embryonic GSH homeostasis could lead to embryotoxicity due to alterations of the critical cellular functions regulated by GSH status. This dissertation investigates the effects of GSH oxidation on embryonic development and describes plausible mechanisms of embryotoxicity resulting from direct oxidation of GSH in organogenesis-stage rat conceptuses in vitro. Diamide (diazenedicarboxylic acid bis-(N, N$\sp\prime$-dimethylamide)) was used as a model compound to evaluate thiol oxidation effects because of its known direct ability to preferentially oxidize GSH to glutathione disulfide (GSSG). Developing rat conceptuses (gestational day 10) exposed to diamide (75-500 $\mu$M) in vitro showed axial rotation defects, embryonic growth retardation, decreased vitelline vasculature, and collapse of the amnion. Concomitant with changes in GSH and GSSG levels, diamide (75-500 $\mu$M) significantly increased protein-glutathione mixed disulfide (protein-S-SG) formation in cultured conceptuses. The enzyme glutathione disulfide reductase, which plays a significant role in maintenance of GSH status and in protection against embryotoxicity induced by diamide, was also found to regulate the formation protein-S-SG in the conceptus. Exposure of the conceptus to diamide (50-500 $\mu$M) resulted in a concentration-dependent stimulation of the pentose phosphate shunt pathway. In contrast, lactate production and activities of the glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GPD) and phosphofructokinase, were significantly decreased in conceptuses exposed to 250-500 $\mu$M diamide. Further experiments demonstrated that GPD may form mixed-disulfides with GSH, leading to enzyme inactivation and inhibition of glycolysis. Because the greater effects of diamide on GSH oxidation, protein-S-SG formation and enzyme inhibition were seen in the visceral yolk sac (VYS), it is likely that, under these experimental conditions, the embryotoxicity of diamide was mediated primarily via altered GSH-regulated cellular functions in the VYS. Mechanisms of embryotoxicity associated with GSH oxidation and protein-S-SG formation by diamide include disrupted conceptal energy production (glycolysis), altered VYS angiogenesis and a collapse of the amnion. Several environmental and therapeutic teratogens/embryotoxins such as phenytoin, paraquat and adriamycin also produce oxidative stress, leading to the net oxidation of GSH. The findings presented in this dissertation suggest that GSH oxidation and protein-S-SG formation per se may be directly involved in the mechanisms of embryotoxicity.